M.George+notebook


 * 11.27.2013 **
 * PROJECT: Zoom 2D gel electrophoresis protocol **


 * Materials **
 * **Name ** || **Quantity ** || **Catalog no. ** ||
 * ZOOM® IPGRunner™ Cassettes || 10 || ZM0003 ||
 * NuPAGE® Novex 4-12% Bis-Tris ZOOM® Gel || 10 gels || NP0330BOX ||
 * ZOOM® Carrier Ampholytes pH 3-10 || 10 ml || ZM0021 ||
 * ZOOM® Strip pH 3-10L (linear) || 12 strips || ZM0018 ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">ZOOM® 2D Protein Solubilizer 1 || <span style="font-family: Arial,Helvetica,sans-serif;">20 ml || <span style="font-family: Arial,Helvetica,sans-serif;">ZS10001 ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">SilverQuest™ Silver Staining Kit || <span style="font-family: Arial,Helvetica,sans-serif;">1 kit || <span style="font-family: Arial,Helvetica,sans-serif;">LC6070 ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">N,N-Dimethylacrylamide (DMA) ||  || <span style="font-family: Arial,Helvetica,sans-serif;">Aldrich, cat. no. 27413-5 ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">Protease Inhibitor tablets ||  || <span style="font-family: Arial,Helvetica,sans-serif;">Roche cat. no. 1873580 ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">1 M Tris Base ||  ||   ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">2 M DTT ||  ||   ||
 * Methods **
 * Methods **
 * Methods **

had trouble with all the pictures making it into this web-space so I saved today's entry into a word document and have made it public through this link:

https://www.dropbox.com/s/n5mp7xmzeuisos8/11_27_lab_entry.docx


 * 11.25.2013 **
 * PROJECT: water chemistry data **

<span style="font-family: Arial,Helvetica,sans-serif;">Tanks labeled from left (by CO2 tank) to right
 * **Tank A/B** || || || || || || || ||
 * **Date Sampled** || **measured pH, NBS scale** || **Temp, °C** || **Salinity, estimated** || **TA (umol/kgSW), estimated** || **pCO2 (uatm), calculated** || **Ω Ca, calculated** || **Ω Ar, calculated** ||
 * 11/8/2013 || 7.50 || 16.60 || 30.00 || 2100.00 || 2045.33 || 0.83 || 0.53 ||
 * 11/11/2013 || 7.72 || 16.40 || 30.00 || 2100.00 || 1198.92 || 1.33 || 0.85 ||
 * 11/12/2013 || 7.56 || 16.30 || 30.00 || 2100.00 || 1765.80 || 0.94 || 0.60 ||
 * 11/13/2013 || 7.53 || 16.50 || 30.00 || 2100.00 || 1901.54 || 0.88 || 0.56 ||
 * 11/15/2013 || 7.49 || 16.60 || 30.00 || 2100.00 || 2094.86 || 0.81 || 0.52 ||
 * 11/18/2013 || 7.54 || 16.70 || 30.00 || 2100.00 || 1860.12 || 0.91 || 0.58 ||
 * 11/25/2013 || 7.52 || 16.40 || 30.00 || 2100.00 || 1945.75 || 0.86 || 0.55 ||
 * 11/27/2013 || 7.54 || 16.60 || 30.00 || 2100.00 || 1858.28 || 0.90 || 0.58 ||


 * **Tank C/D** |||| || || || || || ||
 * **Date Sampled** || **measured pH, NBS scale** || **Temp, °C** || **Salinity, estimated** || **TA (umol/kgSW), estimated** || **pCO2 (uatm), calculated** || **Ω Ca, calculated** || **Ω Ar, calculated** ||
 * 11/8/2013 || 7.84 || 16.60 || 30.00 || 2100.00 || 893.0 || 1.73 || 1.10 ||
 * 11/11/2013 || 7.88 || 16.40 || 30.00 || 2100.00 || 806.6 || 1.87 || 1.19 ||
 * 11/12/2013 || 7.89 || 16.30 || 30.00 || 2100.00 || 786.0 || 1.90 || 1.21 ||
 * 11/13/2013 || 7.87 || 16.50 || 30.00 || 2100.00 || 827.8 || 1.84 || 1.17 ||
 * 11/15/2013 || 7.84 || 16.60 || 30.00 || 2100.00 || 893.0 || 1.73 || 1.10 ||
 * 11/18/2013 || 7.87 || 16.70 || 30.00 || 2100.00 || 829.2 || 1.85 || 1.18 ||
 * 11/25/2013 || 7.81 || 16.40 || 30.00 || 2100.00 || 960.5 || 1.61 || 1.03 ||
 * 11/27/2013 || 7.87 || 16.60 || 30.00 || 2100.00 || 828.5 || 1.84 || 1.17 ||

All values calculated with CO2sys Standards used in calculations:
 * Borate |||||||||| Uppstrom, Deep-Sea Research, 21:161-162, 1974 ||  ||


 * KHSO4 |||||||||||| Dickson, Journal of Chemical Thermodynamics, 22:113-127, 1990. ||


 * K1, K2 |||||||||||| from Mehrbach et al., Limnology and Oceaneanography, 18:897-907, 1973 ||
 * |||||||||||| refit by Dickson and Millero, Deep-Sea Research, 36:983, 1989 ||


 * 11.15.2013 **
 * PROJECT: proposed protocol, adapted from Tomanek et al. 2011, JEB **

<span style="font-family: Arial,Helvetica,sans-serif;">Protocol: <span style="font-family: Arial,Helvetica,sans-serif;">Homogenization <span style="font-family: Arial,Helvetica,sans-serif;">1. Homogenize sample in homogenization buffer* (1:4 ratio sample to buffer) <span style="font-family: Arial,Helvetica,sans-serif;">2. Centrifuge at room temperature for 30 minutes at 16100 g <span style="font-family: Arial,Helvetica,sans-serif;">3. Precipitate supernantant by washing with ice-cold 10% trichloroacetic acid in acetone <span style="font-family: Arial,Helvetica,sans-serif;">4. Store samples overnight –20°C <span style="font-family: Arial,Helvetica,sans-serif;">5. Centrifuge sample at 4°C for 15 minutes at 18000 g <span style="font-family: Arial,Helvetica,sans-serif;">6. Discard supernatant <span style="font-family: Arial,Helvetica,sans-serif;">7. Wash pellet and re-suspend in rehydration buffer* by vortexing for 5 minutes

<span style="font-family: Arial,Helvetica,sans-serif;">Protein Separation: <span style="font-family: Arial,Helvetica,sans-serif;">1. seperate proteins according to isoelectric point (pI) on immobilzed pH gradient (IPG) strips [pH 4–7, 11cm; GE Healthcare] using 400ug of protein per strip

<span style="font-family: Arial,Helvetica,sans-serif;">not sure how to carry out step 2 here: <span style="font-family: Arial,Helvetica,sans-serif;">"The isoelectric focusing protocol started with a passive rehydration step (5hrs), followed by 12hrs of active rehydration (50V) using an isoelectric focusing cell" <span style="font-family: Arial,Helvetica,sans-serif;">500V for 1hr, 1000V for 1hr and 8000V for 2.5hrs (all changes occurred in rapid mode).

<span style="font-family: Arial,Helvetica,sans-serif;">3. Following isoelectric focusing, freeze gels at –80°C.

<span style="font-family: Arial,Helvetica,sans-serif;">2D Gel Electrophoresis: <span style="font-family: Arial,Helvetica,sans-serif;">1. Thaw samples to room temperature <span style="font-family: Arial,Helvetica,sans-serif;">2. Incubate IPG strips in equilibration buffer* for 15 minutes w/ 65 mmol/L dithiothreitol <span style="font-family: Arial,Helvetica,sans-serif;">3. Decant solution <span style="font-family: Arial,Helvetica,sans-serif;">4. Incubate IPG strips in equilibration buffer* for 15 minutes w/ 135 mmol/L iodoacetamide <span style="font-family: Arial,Helvetica,sans-serif;">5. Place IPG strips on top of an 11.8% polyacrylamide gel with a 0.8% agarose solution containing Laemmli SDS electrophoresis buffer* <span style="font-family: Arial,Helvetica,sans-serif;">6. Run gel at 200 V for 55 minutes with a recirculating water bath set at 10°C using Criterion Dodeca cells (Biorad) <span style="font-family: Arial,Helvetica,sans-serif;">7. stain gels with colloidal Coomassie Blue dye (G-250) overnight <span style="font-family: Arial,Helvetica,sans-serif;">8. Destain gels by washing repeatedly with Milli-Q water for at least 48hrs. <span style="font-family: Arial,Helvetica,sans-serif;">9. Scan gel / take picture for analysis

<span style="font-family: Arial,Helvetica,sans-serif;">Reagents: <span style="font-family: Arial,Helvetica,sans-serif;">Homogenization Buffer:
 * <span style="font-family: Arial,Helvetica,sans-serif;">7 mol/L urea
 * <span style="font-family: Arial,Helvetica,sans-serif;">2 mol/L thiourea
 * <span style="font-family: Arial,Helvetica,sans-serif;">23 mmol/L ASB-14 (amidosulfobetaine-14)
 * <span style="font-family: Arial,Helvetica,sans-serif;">40 mmol/L Tris-base
 * <span style="font-family: Arial,Helvetica,sans-serif;">0.5% immobilized pH 4–7 gradient (IPG) buffer (GE Healthcare, Piscataway, NJ, USA)

<span style="font-family: Arial,Helvetica,sans-serif;">Rehydration Buffer:
 * <span style="font-family: Arial,Helvetica,sans-serif;">7 mol/L urea
 * <span style="font-family: Arial,Helvetica,sans-serif;">2 mol/L thiourea
 * <span style="font-family: Arial,Helvetica,sans-serif;">3.25 mmol/L cholamidopropyl-dimethylammonio-propanesulfonic acid (CHAPS)
 * <span style="font-family: Arial,Helvetica,sans-serif;">2% nonyl phenoxylpolyethoxylethanol-40 (NP-40)
 * <span style="font-family: Arial,Helvetica,sans-serif;">0.0005% Bromophenol Blue
 * <span style="font-family: Arial,Helvetica,sans-serif;">0.5% immobilized pH 4–7 gradient (IPG) buffer (GE Healthcare, Piscataway, NJ, USA)
 * <span style="font-family: Arial,Helvetica,sans-serif;">100 mmol/L dithioerythritol

<span style="font-family: Arial,Helvetica,sans-serif;">Equilibration Buffer:
 * <span style="font-family: Arial,Helvetica,sans-serif;">375 mmol/L Tris-base
 * <span style="font-family: Arial,Helvetica,sans-serif;">6 mol/L urea
 * <span style="font-family: Arial,Helvetica,sans-serif;">30% glycerol
 * <span style="font-family: Arial,Helvetica,sans-serif;">2% sodium dodecyl sulfate (SDS)
 * <span style="font-family: Arial,Helvetica,sans-serif;">0.0002% Bromophenol Blue

<span style="font-family: Arial,Helvetica,sans-serif;">Laemmli SDS electrophoresis buffer:
 * <span style="font-family: Arial,Helvetica,sans-serif;">25 mmol/L Tris-base
 * <span style="font-family: Arial,Helvetica,sans-serif;">192 mmol/L glycine
 * <span style="font-family: Arial,Helvetica,sans-serif;">0.1% sodium dodecyl sulfate (SDS)

<span style="font-family: Arial,Helvetica,sans-serif;">Timeline: <span style="font-family: Arial,Helvetica,sans-serif;">11/19: <span style="font-family: Arial,Helvetica,sans-serif;">Make all buffer solutions <span style="font-family: Arial,Helvetica,sans-serif;">11/22: <span style="font-family: Arial,Helvetica,sans-serif;">Practice, find all equipment <span style="font-family: Arial,Helvetica,sans-serif;">11/25: <span style="font-family: Arial,Helvetica,sans-serif;">12pm:4pm - protein homogenization <span style="font-family: Arial,Helvetica,sans-serif;">11/26: <span style="font-family: Arial,Helvetica,sans-serif;">1:30pm:6pm - 2D gel, let stain overnight, let destain over holiday.


 * <span style="font-family: Arial,Helvetica,sans-serif;">11.12.2013 **
 * <span style="font-family: Arial,Helvetica,sans-serif;">PROJECT: update **

<span style="font-family: Arial,Helvetica,sans-serif;">Mussels were sampled today from each treatment. Gill and foot tissue was dissected, labeled as follows, and stored in the -80 freezer.

<span style="font-family: Arial,Helvetica,sans-serif;">

<span style="font-family: Arial,Helvetica,sans-serif;">Treatment tanks were set up today. 4, 55 Gallon garbage cans containing seawater were labeled as follows:
 * <span style="font-family: Arial,Helvetica,sans-serif;">11.6.2013 **
 * <span style="font-family: Arial,Helvetica,sans-serif;">PROJECT: update **

<span style="font-family: Arial,Helvetica,sans-serif;">Tank A - High CO2, no copper <span style="font-family: Arial,Helvetica,sans-serif;">Tank B - High CO2, copper <span style="font-family: Arial,Helvetica,sans-serif;">Tank C - Low CO2, copper <span style="font-family: Arial,Helvetica,sans-serif;">Tank D - Low CO2, no copper

<span style="font-family: Arial,Helvetica,sans-serif;">Garbage cans were bubbled with either ambient air for the low CO2 treatment (pCO2 approx. 350-400) or CO2/air mixture (pCO2 approx. 1400-1600). The CO2/air mixture was obtained using two mass flow controllers, one attached to a CO2 tank while another was allowed to suck ambient air. Each mass flow controller was then attached to a 18 Gal/hr pump which was in turn attached to a venturi injector. The action of the pump caused a negative pressure at the injector which, in turn, sucked the gas mixture into the water column and dispersed it in a cloud of bubbles.

<span style="font-family: Arial,Helvetica,sans-serif;">0.5mg/L of Copper were added into 2 tanks, constituting a 'high' treatment

<span style="font-family: Arial,Helvetica,sans-serif;">The mass flow controllers were set to 1.1 ml/min of CO2 and 1.1 L/min of ambient air. This calculation assumed that ambient air was 400 ppm CO2.

<span style="font-family: Arial,Helvetica,sans-serif;">Desired pCO2 of water = 1500 uatm <span style="font-family: Arial,Helvetica,sans-serif;">1500 ppm of CO2 in air = 0.15% CO2 to AIR ratio <span style="font-family: Arial,Helvetica,sans-serif;">Ambient air source = 400 ppm CO2 --> 0.04 % CO2 <span style="font-family: Arial,Helvetica,sans-serif;">CO2/AIR mixture set to = 1.1 ml/min CO2 / 1000 ml/min AIR --> 0.11 % CO2 <span style="font-family: Arial,Helvetica,sans-serif;">0.11% CO2 + 0.04 --> approx. 0.15% or 1500 uatm at sea level

<span style="font-family: Arial,Helvetica,sans-serif;">11.5.2013 <span style="font-family: Arial,Helvetica,sans-serif;">PROJECT: update <span style="font-family: Arial,Helvetica,sans-serif;">Mussels were sampled today as a "time zero" or starting condition. Gill and foot tissue was dissected, labeled with the date and the tissue type, and stored in the -80 freezer. <span style="font-family: Arial,Helvetica,sans-serif;">From Diana: Labeled: date, M1 or M2 (depending on which mussel they came from) and mass of tissue

<span style="font-family: Arial,Helvetica,sans-serif;">10.29.2013 <span style="font-family: Arial,Helvetica,sans-serif;">PROJECT: planning

<span style="font-family: Arial,Helvetica,sans-serif;">Summary <span style="font-family: Arial,Helvetica,sans-serif;">Met with lab members and finished experimental design for our mussel project. Seawater, primers, and mussels are not yet available. Will update when project moves forward.

<span style="font-family: Arial,Helvetica,sans-serif;">Lab 4: protein SDS-PAGE and western blot
 * <span style="font-family: Arial,Helvetica,sans-serif;">10.22.2013 **

<span style="font-family: Arial,Helvetica,sans-serif;">Summary <span style="font-family: Arial,Helvetica,sans-serif;">A western blot was run using protein samples that were prepared last week (procedure will be included in today's entry for continuity). cDNA from last week which was amplified with qPCR was visualized using SDS-PAGE electrophoresis and a black light. cDNA results will be added to last weeks lab entry for continuity.

<span style="font-family: Arial,Helvetica,sans-serif;">Notes <span style="font-family: Arial,Helvetica,sans-serif;">My protein of interest was labeled MGEO and was placed in lane 8 of the well of the SDS-PAGE gel. The protein was mantle tissue, from an Olympia oyster (labeled 69) - unknown weight of sample.

<span style="font-family: Arial,Helvetica,sans-serif;">Materials and Methods <span style="font-family: Arial,Helvetica,sans-serif;">Supplies and Reagents
 * <span style="font-family: Arial,Helvetica,sans-serif;">heating block with water bath
 * <span style="font-family: Arial,Helvetica,sans-serif;">platform rocker/shaker
 * <span style="font-family: Arial,Helvetica,sans-serif;">Primary Antibody Solution
 * <span style="font-family: Arial,Helvetica,sans-serif;">Blocking Solution
 * <span style="font-family: Arial,Helvetica,sans-serif;">Antibody Wash
 * <span style="font-family: Arial,Helvetica,sans-serif;">Secondary Antibody Solution
 * <span style="font-family: Arial,Helvetica,sans-serif;">Chromogenic Substrate
 * <span style="font-family: Arial,Helvetica,sans-serif;">SDS-PAGE gel
 * <span style="font-family: Arial,Helvetica,sans-serif;">2X SDS reducing sample buffer
 * <span style="font-family: Arial,Helvetica,sans-serif;">gel running buffer
 * <span style="font-family: Arial,Helvetica,sans-serif;">light box
 * <span style="font-family: Arial,Helvetica,sans-serif;">Tris-Glycine transfer buffer
 * <span style="font-family: Arial,Helvetica,sans-serif;">filter paper
 * <span style="font-family: Arial,Helvetica,sans-serif;">nitrocellulose membrane
 * <span style="font-family: Arial,Helvetica,sans-serif;">semi-dry transfer station

<span style="font-family: Arial,Helvetica,sans-serif;">(1) PROTEIN EXTRACTION PROTOCOL
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Record the weight of your tissue that has been denoted on the tube.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Label the snap cap tube containing your tissue sample with your initials and the date using a lab marker.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Add 500 ul of CellLytic MT solution to the 1.5mL snap cap tube containing your cut piece of frozen tissue.
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Homogenize the tissue with a sterile disposable pestle.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Close the tube and invert the tube several times.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">Please find a few other people at or near this same stage and form a group for this step. Spin the tube in a refrigerated microfuge for 10 mins at max speed.
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">While spinning, label a fresh tube with the word "Protein", source organism/tissue, your initials, and today's date.
 * 8) <span style="font-family: Arial,Helvetica,sans-serif;">Carefully transfer supernatant (the clearish liquid on top) to labeled tube and store tube on ice.

<span style="font-family: Arial,Helvetica,sans-serif;">(2) SDS-PAGE PROTOCOL
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Begin boiling water on hot plate.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">In a fresh, 1.5mL SCREW CAP tube add 15uL of your protein stock and 15uL of 2X Reducing Sample Buffer. Return your protein stock to the box in the -20C freezer labeled protein samples.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Mix sample by flicking. Briefly centrifuge (10s) to pool liquid in bottom of tube.
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Boil sample for 5 mins.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">While sample is boiling, observe assembly of gel box and gels. Rinse gel wells thoroughly as demonstrated.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">When sample is finished boiling, immediately centrifuge for 1min. to pool liquid.
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">Slowly load your entire sample into the appropriate well using a gel loading tip.
 * 8) <span style="font-family: Arial,Helvetica,sans-serif;">Put lid on gel box and plug electrodes into appropriate receptacles on the power supply.
 * 9) <span style="font-family: Arial,Helvetica,sans-serif;">Turn power supply on and set voltage to 150V. Run for 45mins. CHECK YOUR AGAROSE GEL RESULTS. MAKE SURE EVERYTHING IS SET UP FOR WESTERN BLOT.
 * 10) <span style="font-family: Arial,Helvetica,sans-serif;">Turn off power supply and disconnect gel box from power supply.
 * 11) <span style="font-family: Arial,Helvetica,sans-serif;">Remove lid from gel box.
 * 12) <span style="font-family: Arial,Helvetica,sans-serif;">Disengage the tension wedge.
 * 13) <span style="font-family: Arial,Helvetica,sans-serif;">Remove gel from gel box.
 * 14) <span style="font-family: Arial,Helvetica,sans-serif;">Carefully crack open cassette to expose gel.
 * 15) <span style="font-family: Arial,Helvetica,sans-serif;">Trim wells at top of gel.
 * 16) <span style="font-family: Arial,Helvetica,sans-serif;">Notch a designated corner of the gel to help you remember the correct orientation of the gel (i.e. which is the top/bottom of the gel, which is the right/left side(s) of the gel)
 * 17) <span style="font-family: Arial,Helvetica,sans-serif;">Proceed to Western Blotting protocol.

<span style="font-family: Arial,Helvetica,sans-serif;">(3) WESTERNBREEZE CHROMOGENIC WESTERN BLOT IMMUNODETECTION
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Soak the filter paper, membrane and gel in Tris-Glycine Transfer Buffer for 15 minutes.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Assemble the blotting sandwich in the semi-dry blotting appartus:Transfer the blot for 30 minutes at 20V
 * 3) Anode (+++)
 * 4) filter paper
 * 5) membrane
 * 6) gel
 * 7) filter paper
 * 8) cathode (---)
 * 9) <span style="font-family: Arial,Helvetica,sans-serif;">Remove the gel from the sandwich and rinse off adhering pieces of gel with transfer buffer.
 * 10) <span style="font-family: Arial,Helvetica,sans-serif;">Wash membrane 2 times, for 5 minutes each, with 20 mL of pure water.
 * 11) <span style="font-family: Arial,Helvetica,sans-serif;">Put the membrane in the plastic box and add 10 mL of Blocking Solution. Cover and incubate overnight on a rotary shaker set at 1 revolution/second. (This was done for 30 minutes, not overnight in lab!!)
 * 12) <span style="font-family: Arial,Helvetica,sans-serif;">Your TA will do the rest of the steps. After class tomorrow you can come and see your results.
 * 13) <span style="font-family: Arial,Helvetica,sans-serif;">Decant liquid.
 * 14) <span style="font-family: Arial,Helvetica,sans-serif;">Rinse the membrane with 20 mL of water for 5 minutes, then decant. Repeat.
 * 15) <span style="font-family: Arial,Helvetica,sans-serif;">Incubate the membrane in 10 mL of Primary Antibody Solution. Decant the solution.
 * 16) <span style="font-family: Arial,Helvetica,sans-serif;">Rinse the membrane with 20 mL of Antibody Wash for 5 minutes, then decant. Repeat 3 times.
 * 17) <span style="font-family: Arial,Helvetica,sans-serif;">Incubate the membrane in 10 mL of Secondary Antibody Solution for 30 minutes. Decant.
 * 18) <span style="font-family: Arial,Helvetica,sans-serif;">Wash the membrane for 5 minutes with 20 mL of Antibody wash, then decant. Repeat 3 times.
 * 19) <span style="font-family: Arial,Helvetica,sans-serif;">Rinse the membrane with 20 mL of pure water for 2 minutes, then decant. Repeat twice.
 * 20) <span style="font-family: Arial,Helvetica,sans-serif;">Incubate the membrane in 5 mL of Chromogenic Substrate until a purple band appears. This will occur between 1-60 minutes after adding the Chromogenic Substrate.
 * 21) <span style="font-family: Arial,Helvetica,sans-serif;">Dry the membrane on a clean piece of filter paper to the open air.
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Dry the membrane on a clean piece of filter paper to the open air.

<span style="font-family: Arial,Helvetica,sans-serif;">Results <span style="font-family: Arial,Helvetica,sans-serif;">Results of SDS-PAGE and qPCR were added to last weeks lab manual entry!!!

<span style="font-family: Arial,Helvetica,sans-serif;">Here are the results from the western blot. My sample is in lane 8. Lane one is a ladder. <span style="font-family: Arial,Helvetica,sans-serif;">

<span style="font-family: Arial,Helvetica,sans-serif;">Conclusions <span style="font-family: Arial,Helvetica,sans-serif;">Looking at the results of the western blot is looks like the presence of HSP is evident by a purple band that is present in all samples tested. The band is fairly weak, implying that much protein was not present. It would have been nice if the ladder was still present so I could interpret the band and see if it corresponds in length to a known HSP. Perhaps when I see it next week.

<span style="font-family: Arial,Helvetica,sans-serif;">Reflection <span style="font-family: Arial,Helvetica,sans-serif;">The purpose of this weeks lab was to familiarize us with the SDS-PAGE protocol and to isolate proteins and run a western blot to confirm the present of a target protein. These procedures could be used to measure changes in the amount and type of protein expression in tissues and organisms experiencing some kind of environmental stress. Nothing about this week remains unclear, the procedure and supporting material was really helpful.

<span style="font-family: Arial,Helvetica,sans-serif;">10.21.2013 <span style="font-family: Arial,Helvetica,sans-serif;">How to design a primer <span style="font-family: Arial,Helvetica,sans-serif;">Primers, or oligonucleotides (oligos), are short stretches of synthetic DNA that are used most commonly for PCR and DNA sequencing. They direct DNA polymerases to specific regions on larger DNA molecules for amplification. They are designed in pairs to amplify DNA in the forward and reverse directions. Oligos are custom synthesized by various manufacturer's to contain the precise sequence requested by the customer. For a good introduction to the theory of primer design proceed to this [|link].

<span style="font-family: Arial,Helvetica,sans-serif;">Here is a brief list of things to take into consideration when designing primers. Although none of these are absolute, they will help ensure your primers will hybridize to your target sequence with the best efficiency.

<span style="font-family: Arial,Helvetica,sans-serif;">1. Design your primers to be within 18-30 bases in length. <span style="font-family: Arial,Helvetica,sans-serif;">2. The melting temperature (Tm) of primers should be within 2C of each other. <span style="font-family: Arial,Helvetica,sans-serif;">3. Avoid primer dimers and primer hairpins <span style="font-family: Arial,Helvetica,sans-serif;">4. Avoid high G/C stretches, particularly at the 3' end <span style="font-family: Arial,Helvetica,sans-serif;">5. G/C clamp at 3' end of primers.

<span style="font-family: Arial,Helvetica,sans-serif;">Primer design is most commonly done via computerized means and the algorithms used take the above rules into consideration. Of course, the user always has the opportunity to adjust the parameters that define how primers are designed by the software. There is a great deal of software available for primer design. Two commonly used primer design tools are [|NCBI Primer] and [|Primer 3]. The software will allow you to enter a full DNA sequence and then define what region(s) you would like to amplify, the ideal size of the amplicon (PCR product), the ideal length of the oligos, etc. However, often the preset conditions are already optmized to Additionally, after you have selected some proposed primers, you can compare melting temps, G/C content, primer dimer/hairpin probabilitites, etc.

<span style="font-family: Arial,Helvetica,sans-serif;">We will go over the process of designing primers in more detail in lab.

<span style="font-family: Arial,Helvetica,sans-serif;">10.21.2013 <span style="font-family: Arial,Helvetica,sans-serif;">How to make an Agarose gel:

<span style="font-family: Arial,Helvetica,sans-serif;">Supplies and Equipment:
 * <span style="font-family: Arial,Helvetica,sans-serif;">1L flask
 * <span style="font-family: Arial,Helvetica,sans-serif;">agarose
 * <span style="font-family: Arial,Helvetica,sans-serif;">1X TAE
 * <span style="font-family: Arial,Helvetica,sans-serif;">Ethidium bromide
 * <span style="font-family: Arial,Helvetica,sans-serif;">Microwave
 * <span style="font-family: Arial,Helvetica,sans-serif;">Gel rigs

<span style="font-family: Arial,Helvetica,sans-serif;">AGAROSE GEL POURING PROCEDURE
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Weigh 2g of agarose and mix with 150mL 1x TAE in a 1L flask
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Microwave solution for ~ 3 minutes. Keep an eye on the solution so that it does not boil over. You want the solution to be clear - no precipitate and no bubbles.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Cool solution (you should be able to touch the flask for a few seconds), then add 12uL ethidium bromide(EtBr). WARNING: EtBr is a carcinogen be sure to wear gloves and appropriately dispose tip waste.
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Mix thoroughly by swirling, then pour into gel tray.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Add gel combs. Using a clean pipet tip, pop any bubbles that could get in the way of your PCR product.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">After gel is set, wrap in plastic wrap (label with your initials and date) and place gel in the fridge if not using immediately.

<span style="font-family: Arial,Helvetica,sans-serif;">10.15.2013 <span style="font-family: Arial,Helvetica,sans-serif;">Lab 3: Quantitative PCR

<span style="font-family: Arial,Helvetica,sans-serif;">Summary <span style="font-family: Arial,Helvetica,sans-serif;">Using RNA isolated last week cDNA was produced using a reverse transcriptase protocol. Primers for a region of interest were added to the mixture and quantitative PCR was run on the cDNA sample to look for the presence of HIF in a sample of oyster mantle tissue. Plating on an agarose gel will be done next week. Protein extraction and primer design will also take place next week.

<span style="font-family: Arial,Helvetica,sans-serif;">Materials and Methods <span style="font-family: Arial,Helvetica,sans-serif;">Supplies and Equipment:
 * <span style="font-family: Arial,Helvetica,sans-serif;">PCR Plates (white); optically clear caps
 * <span style="font-family: Arial,Helvetica,sans-serif;">1.5 ml microfuge tubes (RNAse free)
 * <span style="font-family: Arial,Helvetica,sans-serif;">Nuclease Free water
 * <span style="font-family: Arial,Helvetica,sans-serif;">Opticon thermal cycler
 * <span style="font-family: Arial,Helvetica,sans-serif;">2x Immomix Master Mix

<span style="font-family: Arial,Helvetica,sans-serif;">(1) qPCR PROCEDURE

<span style="font-family: Arial,Helvetica,sans-serif;">You will run each template (cDNA) in duplicate in addition to two negative controls (no template) - calculate how many reactions this will be!
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Prepare master mix: Prepare enough master mix for your number of reactions +1 to ensure sufficient volume recovery.

<span style="font-family: Arial,Helvetica,sans-serif;">For a 25μl reaction volume:
 * <span style="font-family: Arial,Helvetica,sans-serif;">Component || <span style="font-family: Arial,Helvetica,sans-serif;">Volume || <span style="font-family: Arial,Helvetica,sans-serif;">Final Conc. ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">Master Mix (SsoFast EvaGreen supermix) || <span style="font-family: Arial,Helvetica,sans-serif;">12.5µL || <span style="font-family: Arial,Helvetica,sans-serif;">1x ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">upstream primer, 10μM || <span style="font-family: Arial,Helvetica,sans-serif;">0.5μl || <span style="font-family: Arial,Helvetica,sans-serif;">2.5μM ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">downstream primer, 10μM || <span style="font-family: Arial,Helvetica,sans-serif;">0.5μl || <span style="font-family: Arial,Helvetica,sans-serif;">2.5μM ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">Ultra Pure Water || <span style="font-family: Arial,Helvetica,sans-serif;">10.5uL || <span style="font-family: Arial,Helvetica,sans-serif;">NA ||


 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Add mastermix to wells of a white PCR plate
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Thaw cDNA samples.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Add 1uL cDNA template to each reaction.
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Add 1uL of ultra pure water to the negative control wells.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Cap the wells securely.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">If necessary, spin the strips to collect volume in the bottom of the wells.
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">Ensure the lids are clean and place strips on ice. (I like to wipe the lids with a clean kimwipe)
 * 8) <span style="font-family: Arial,Helvetica,sans-serif;">Load the plate, verify the PCR conditions and start the run (this will be done by your TA).

<span style="font-family: Arial,Helvetica,sans-serif;">PCR conditions:
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">95°C for 10 minutes
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">95°C for 15s
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">55 °C for 15 s
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">72°C for 15 s (+ plate read)
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Return to step 2 39 more times
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">95°C for 10s
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)

<span style="font-family: Arial,Helvetica,sans-serif;">Results <span style="font-family: Arial,Helvetica,sans-serif;">qPCR samples were left to run overnight. Results will be obtained next week.

<span style="font-family: Arial,Helvetica,sans-serif;">UPDATE: Here are the results from the qPCR. My samples were in lanes H1-H4, with lanes 1 and 2 containing cDNA.

<span style="font-family: Arial,Helvetica,sans-serif;">
 * <span style="font-family: Arial,Helvetica,sans-serif;">H1 || <span style="font-family: Arial,Helvetica,sans-serif;">SBGnew || <span style="font-family: Arial,Helvetica,sans-serif;">Sample ||  || <span style="font-family: Arial,Helvetica,sans-serif;">N/A || <span style="font-family: Arial,Helvetica,sans-serif;">N/A ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">H2 || <span style="font-family: Arial,Helvetica,sans-serif;">SBGnew || <span style="font-family: Arial,Helvetica,sans-serif;">Sample ||  || <span style="font-family: Arial,Helvetica,sans-serif;">N/A || <span style="font-family: Arial,Helvetica,sans-serif;">N/A ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">H3 || <span style="font-family: Arial,Helvetica,sans-serif;">SBGnew || <span style="font-family: Arial,Helvetica,sans-serif;">Sample ||  || <span style="font-family: Arial,Helvetica,sans-serif;">N/A || <span style="font-family: Arial,Helvetica,sans-serif;">N/A ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">H4 || <span style="font-family: Arial,Helvetica,sans-serif;">SBGnew || <span style="font-family: Arial,Helvetica,sans-serif;">Sample ||  || <span style="font-family: Arial,Helvetica,sans-serif;">N/A || <span style="font-family: Arial,Helvetica,sans-serif;">N/A ||

<span style="font-family: Arial,Helvetica,sans-serif;">UPDATE: Here is the gel from the next weeks lab. My sample is in lab 8.

<span style="font-family: Arial,Helvetica,sans-serif;">

<span style="font-family: Arial,Helvetica,sans-serif;">Conclusions <span style="font-family: Arial,Helvetica,sans-serif;">No conclusions can be drawn as yet. This section will be filled in next week.

<span style="font-family: Arial,Helvetica,sans-serif;">UPDATE: both quantification and visualization using gel electrophoresis show that no cDNA was amplified in any of my samples.

<span style="font-family: Arial,Helvetica,sans-serif;">Reflection <span style="font-family: Arial,Helvetica,sans-serif;">The purpose of this lab was to familiarize us with the procedure for producing cDNA, adding primers, and running qPCR. qPCR amplifies a cDNA region of interest for use in other protocols. qPCR is also a key component used in sequencing genomes. Nothing at this point is unclear or needs more information. I will probably be confused next week with protein extraction so stay tuned.

<span style="font-family: Arial,Helvetica,sans-serif;">UPDATE: cDNA in my samples were either not present of not amplified. It remain unclear as to what actually occurred. Another possibility is that the HIF was not present in my samples, which seems more likely than HSP not being present, as hypoxia is not always a source of stress in marine systems but can be largely seasonal.

<span style="font-family: Arial,Helvetica,sans-serif;">10.8.2013 <span style="font-family: Arial,Helvetica,sans-serif;">Lab 2: RNA isolation part 2

<span style="font-family: Arial,Helvetica,sans-serif;">Summary <span style="font-family: Arial,Helvetica,sans-serif;">The RNA isolation procedure that was started during last weeks lab will was completed. The procedure to produce cDNA from our RNA was also started with the goal of running PCR next week. Members of the lab brain-stormed regarding projects including organisms of interest, possible stresses, and what responses to look for.

<span style="font-family: Arial,Helvetica,sans-serif;">Notes
 * <span style="font-family: Arial,Helvetica,sans-serif;">RNA degrades quickly, cDNA lasts longer and is useful for PCR
 * <span style="font-family: Arial,Helvetica,sans-serif;">PCR amplifies your DNA by a factor of 2 each run
 * <span style="font-family: Arial,Helvetica,sans-serif;">To make cDNA you need
 * RNA
 * nucleotides
 * an enzyme to carry out the reaction (reverse transcriptase)
 * Buffer

<span style="font-family: Arial,Helvetica,sans-serif;">Material and Methods <span style="font-family: Arial,Helvetica,sans-serif;">Samples <span style="font-family: Arial,Helvetica,sans-serif;">All of my samples were labeled with a big MGEO. The only product from today's lab was a vial of cDNA which will be used next week for PCR.

<span style="font-family: Arial,Helvetica,sans-serif;">RNA extraction protocol continued from Lab 1 <span style="font-family: Arial,Helvetica,sans-serif;">Turn on heating block to 55°C.
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Incubate your homogenized tissue sample (from Lab 1) tube at room temperature (RT) for 5 mins.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">In the fume hood, add 200uL of chloroform to your sample and close the tube. NOTE: Due to the high volatility of chloroform, pipetting needs to be done carefully and quickly. Have your tube open and close to the container of chloroform before drawing and chloroform into your pipette tip.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Vortex vigorously for 30s. You are vortexing correctly if the solution becomes a milky emulsion.
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Incubate tube at RT for 5 mins.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Spin tube in refrigerated microfuge for 15 mins. @ max speed.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">Gently remove tube from microfuge. Be sure not to disturb the tube.
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">Slowly and carefully transfer most of the aqueous phase (the top, clear portion) to a fresh microfuge tube (YOU WANT THIS!!!!!!). Do NOT transfer ANY of the interphase (the white, cell debris between the aqueous and organic phase) <-- this is garbage and should be pink

<span style="font-family: Arial,Helvetica,sans-serif;">NOTE: during this lab my pink aqueous and organic phase was on the top!!!!!


 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Close the tube containing the organic and interphase and properly dispose of the liquid inside the tube as well as the tube itself at the end of the lab.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Add 500uL isopropanol to the new tube containing your RNA and close the tube.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Mix by inverting the tube numerous times until the solution appears uniform. Pay particular attention to the appearance of the solution along the edge of the tube. If mixed properly, it should no longer appear viscous/"lumpy".
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Incubate at RT for 10 mins.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Spin in refrigerated microfuge at max speed for 8 mins. When placing your tube in the microfuge position the tube hinge pointing up, away from the center of the microfuge.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">A small, white pellet (RNA and salts) should be present. If not, do not fret an continue with the procedure.
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">Remove supernatant < LIQUID PHASE! >
 * 8) <span style="font-family: Arial,Helvetica,sans-serif;">Add 1mL of 75% EtOH to pellet. Close tube and vortex briefly to dislodge pellet from the side of the tube. If the pellet does not become dislodged, that is OK.
 * 9) <span style="font-family: Arial,Helvetica,sans-serif;">Spin in refrigerated microfuge at 7500g for 5mins.
 * 10) <span style="font-family: Arial,Helvetica,sans-serif;">Carefully remove supernatant. Pellet may be very loose. Make sure not to remove pellet!
 * 11) <span style="font-family: Arial,Helvetica,sans-serif;">Briefly spin tube (~15s) to pool residual EtOH.
 * 12) <span style="font-family: Arial,Helvetica,sans-serif;">Using a small pipette tip (P10 or P20 tips), remove remaining EtOH.
 * 13) <span style="font-family: Arial,Helvetica,sans-serif;">Leave tube open and allow pellet to dry at RT for no more than 5mins.
 * 14) <span style="font-family: Arial,Helvetica,sans-serif;">Resuspend pellet in 100uL of 0.1%DEPC-H2O by pipetting up and down until pellet is dissolved.
 * 15) <span style="font-family: Arial,Helvetica,sans-serif;">Incubated tube at 55C for 5mins. to help solubilize RNA.
 * 16) <span style="font-family: Arial,Helvetica,sans-serif;">Remove tube from heat, flick a few times to mix and place sample on ice. This will be your stock RNA sample.
 * 17) <span style="font-family: Arial,Helvetica,sans-serif;">Quantitate RNA yield using Nanodrop spectrophotometer

<span style="font-family: Arial,Helvetica,sans-serif;">RNA Quantification <span style="font-family: Arial,Helvetica,sans-serif;">NOTE: Always keep your RNA samples on ice!
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Click "Blank", to zero the instrument. NOTE: steps 1 and 2 only need to be done once for the whole class.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Pipette 2µL of your RNA sample onto the Nanodrop pedestal and lower the arm
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Click "Measure". Record your RNA concentration (ng/µL), A260/280 ratio and A260/230 ratio. NOTE: The Nanodrop uses the Beer-Lambert Law to calculate RNA concentration for you. See Lab 1 notes on RNA extraction for more information on the calculation and how to evaluate RNA purity using A260/280 and A260/A230 ratios.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Raise the arm and wipe off you sample with a KimWipe
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">Clearly label your stock RNA sample with the word "RNA", source organism/tissue, your initials, today's date and the concentration in ug/uL.
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">Give your samples to the TA for storage at -80C.

<span style="font-family: Arial,Helvetica,sans-serif;">cDNA: reverse transcription protocol
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Mix your stock RNA sample by inverting tube several times.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">In a 0.5 ml PCR tube labeled with your initials and “cDNA” combine the following:
 * 3) 5 μl of YOUR total RNA (extracted and quantified in lab)
 * 4) 1 μl of oligo dT
 * 5) 4 μl of nuclease free H2O
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">Incubate the mixture for 5 min at 70°C on the thermocycler then immediately transfer to ice. Briefly centrifuge you tube and the add the following:Incubate the mixture for 60 min at 42°C and then heat inactivate at 70°C for 3 min on the thermocycler.Spin down the sample in a desk top centrifuge.
 * 7) 5 μl of M-MLV 5X Reaction Buffer
 * 8) 5 ul of dNTPs
 * 9) 1 μl of M-MLV RT
 * 10) 4 μl of nuclease free H2O
 * 11) <span style="font-family: Arial,Helvetica,sans-serif;">Store on ice or at -20°C
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Store on ice or at -20°C

<span style="font-family: Arial,Helvetica,sans-serif;">Results <span style="font-family: Arial,Helvetica,sans-serif;">Purified RNA target A260/280: 1.8 - 2.0 <span style="font-family: Arial,Helvetica,sans-serif;">Purified RNA target A260/230: 1.5 - 2.0

<span style="font-family: Arial,Helvetica,sans-serif;">Nanodrop results: <span style="font-family: Arial,Helvetica,sans-serif;">A260/280: 1.93 <span style="font-family: Arial,Helvetica,sans-serif;">A260/230: 2.34 <span style="font-family: Arial,Helvetica,sans-serif;">Abs: 14.535 <span style="font-family: Arial,Helvetica,sans-serif;">A-260: 33.960 <span style="font-family: Arial,Helvetica,sans-serif;">A-280: 17.576

<span style="font-family: Arial,Helvetica,sans-serif;">Conclusions <span style="font-family: Arial,Helvetica,sans-serif;">Looking at the nanodrop results my RNA A260/280 was right on while my A260/230 was off. I am a little confused if this means. The manual for the nanodrop states the acceptable range for both ratios but doesn't comment if they both have to be perfect. I will ask about this next lab. If it is the case that both ratio needs to be perfect I would not use this RNA for an experiment. My A260/230 being high may be ethanol contamination or the pellet was not properly dissolved.

<span style="font-family: Arial,Helvetica,sans-serif;">Reflection <span style="font-family: Arial,Helvetica,sans-serif;">The purpose of the lab was to gain experience with RNA isolation and how to make cDNA. The procedures outlined in lab today are used to measure gene expression which makes it useful in studies that investigate how stresses that are imposed on an organism are mediated by different genes. What is unclear about the procedure, as outlined in my conclusions, is what happens when my A260/230 is not perfect. I wish their was more information on what these numbers actually mean and when it is OK to continue with a sample and when it is advisable to start over.

<span style="font-family: Arial,Helvetica,sans-serif;">10.1.2013 <span style="font-family: Arial,Helvetica,sans-serif;">Lab 1: DNA isolation; initiate RNA isolation

<span style="font-family: Arial,Helvetica,sans-serif;">Homework <span style="font-family: Arial,Helvetica,sans-serif;">Some of my favorite examples of genes come from drosophila and have really interesting names. One is virilizer which is responsible for a trans-membrane protein that is involved in Sex lethal splicing. Another is wingless which, as you might expect, produces wingless flies when silenced and it a part of the wnt pathway. A third gene is bag of marbles which codes for a protein which functions as a translational repressor by interfering with translation initiation. Not quite sure why it is called bag of marbles though: http://www.sdbonline.org/fly/cytoskel/bagomb1.htm

<span style="font-family: Arial,Helvetica,sans-serif;">Summary <span style="font-family: Arial,Helvetica,sans-serif;">A RNA isolation TriReagent procedure was started using mantle tissue from a pacific oyster with the aim of finishing the procedure next week. DNA isolation (DNazol) and quantification (Nanodrop spec) was performed following a TriReagent protocol and using gill tissue from a pacific oyster. DNA purity was determined using a spectrophotometer.

<span style="font-family: Arial,Helvetica,sans-serif;">Notes
 * <span style="font-family: Arial,Helvetica,sans-serif;">For the MCRgene procedure used in lab today the recommended samples sizes are 25-50 mg of tissue for DNA extractions and 50-100 mg of tissue for RNA extractions.
 * <span style="font-family: Arial,Helvetica,sans-serif;">all 1mL of TriReagent was added to the RNA isolation sample.

<span style="font-family: Arial,Helvetica,sans-serif;">Materials and Methods <span style="font-family: Arial,Helvetica,sans-serif;">samples
 * ~ <span style="font-family: Arial,Helvetica,sans-serif;">Lab Equipment ||~ <span style="font-family: Arial,Helvetica,sans-serif;">Reagents Used ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">micropipettes || <span style="font-family: Arial,Helvetica,sans-serif;">TriReagent (RNA) ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">microfuge tubes || <span style="font-family: Arial,Helvetica,sans-serif;">DNazol (DNA) ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">centrifuge || <span style="font-family: Arial,Helvetica,sans-serif;">Ethanol ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">disposable pestles || <span style="font-family: Arial,Helvetica,sans-serif;">0.1% DEPC water ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">vortexer ||  ||
 * <span style="font-family: Arial,Helvetica,sans-serif;">Oly = olympia oyster
 * <span style="font-family: Arial,Helvetica,sans-serif;">PAC = pacific oyster
 * <span style="font-family: Arial,Helvetica,sans-serif;">g = gill tissue
 * <span style="font-family: Arial,Helvetica,sans-serif;">m = mantle tissue

<span style="font-family: Arial,Helvetica,sans-serif;">A sample of mantle tissue (77 grams) from an Olympia oyster was used for RNA isolation. The microfuge tube containing the sample was labeled with the weight (77), the tissue type (m), the procedure type (RNA), the species (oly), and the operators initials (MGEO). A sample of gill tissue (40 grams) from a Pacific Oyster was used for DNA isolation and quantification. The microfuge tube containing the sample was labeled with the original weight of the sample (40), tissue type (g), species designation (Pac 35c), date collected 10/23/12, the procedure type (DNA), and operator's initials (MGEO).

<span style="font-family: Arial,Helvetica,sans-serif;">For both DNA and RNA extraction an adapted version of the MRCgene manual protocol (http://www.mrcgene.com/tri.htm) was used. The adapted procedure can be found here: <span style="font-family: Arial,Helvetica,sans-serif;">http://genefish.wikispaces.com/441_Lab+1_2013

<span style="font-family: Arial,Helvetica,sans-serif;">RNA isolation protocol
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Label the snap cap tube containing your tissue sample with your initials and the date using a lab marker. Keep the sample stored on ice until you are ready for homogenization.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Add 500uL of TriReagent to the 1.5mL snap cap tube containing your tissue. Store on ice.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Carefully homogenize the tissue using a disposable pestle. If the tissue is difficult to homogenize, carefully close the tube tightly and briefly vortex the sample.
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">After the sample is completely homogenized, add an additional 500uL of TriReagent to the tube and close the tube tightly.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Vortex vigorously for 15s.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">Stop here for Lab 1 and give your labeled homogenized tissue sample to the TA for storage at -80ºC. You will be finishing your RNA extraction in lab next week.

<span style="font-family: Arial,Helvetica,sans-serif;">DNA isolation protocol
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Using a sterile pestle, homogenize your tissue sample in 0.5 mL of DNazol in a 1.5 mL sterile microfuge tube. After the tissue is homogenized, add 0.5 mL more of DNazol and mix well.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Let your sample incubate for 5 minutes at room temperature.
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Spin your sample at 10,000 x g (room temp) for 10 minutes.
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Transfer your supernatant to a new, labeled tube.
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Add 0.5 mL of 100 % ethanol to your sample.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">Mix your sample by inverting your tube 5-8 times.
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">Store your sample at room temperature for 1 minute.
 * 8) <span style="font-family: Arial,Helvetica,sans-serif;">Your DNA should form a cloudy precipitate. Remove the DNA and put in a new tube using your pipette.
 * 9) <span style="font-family: Arial,Helvetica,sans-serif;">Let your sample sit at room temp for 1 minute and remove the rest of the lysate (liquid that is not DNA).
 * 10) <span style="font-family: Arial,Helvetica,sans-serif;">Wash your DNA with 1 mL of 75% ethanol: Pipette the ethanol into your DNA tube, invert 6 times, and let sit for 1 minute. Remove the ethanol from the tube and repeat.
 * 11) <span style="font-family: Arial,Helvetica,sans-serif;">If there is ethanol left at the bottom of your tube after the second wash, remove with a small pipette.
 * 12) <span style="font-family: Arial,Helvetica,sans-serif;">Add 300 µL of 0.1% DEPC water to your DNA and pipette up and down multiple times to dissolve.
 * 13) <span style="font-family: Arial,Helvetica,sans-serif;">Bring your DNA sample up to the Nanodrop to quantify.

<span style="font-family: Arial,Helvetica,sans-serif;">DNA quantification protocol
 * 1) <span style="font-family: Arial,Helvetica,sans-serif;">Pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm.
 * 2) <span style="font-family: Arial,Helvetica,sans-serif;">Select "dsDNA" from the pulldown menu
 * 3) <span style="font-family: Arial,Helvetica,sans-serif;">Click "Blank", to zero the instrument. NOTE: steps 1 and 2 only need to be done once for the whole class.
 * 4) <span style="font-family: Arial,Helvetica,sans-serif;">Pipette 2µL of your DNA sample onto the Nanodrop pedestal and lower the arm
 * 5) <span style="font-family: Arial,Helvetica,sans-serif;">Click "Measure". Record your DNA concentration (ng/µL), A260/280 ratio and A260/230 ratio. NOTE: The Nanodrop uses the Beer-Lambert Law to calculate DNA concentration for you.
 * 6) <span style="font-family: Arial,Helvetica,sans-serif;">Raise the arm and wipe off you sample with a KimWipe
 * 7) <span style="font-family: Arial,Helvetica,sans-serif;">Clearly label your stock DNA sample with the word "DNA", source organism/tissue, your initials, today's date and the concentration in ug/uL.
 * 8) <span style="font-family: Arial,Helvetica,sans-serif;">Store sample at -20ºC.

<span style="font-family: Arial,Helvetica,sans-serif;">notable deviations from the procedure:
 * <span style="font-family: Arial,Helvetica,sans-serif;">step 7 of the DNazol extraction protocol: after this step the DNA/ethanol mixture was spun using a centrifuge on 10000x for 2 minutes. This resulted in a pellet of DNA on the bottom of the microfuge tube.
 * <span style="font-family: Arial,Helvetica,sans-serif;">step 10 of the DNazol extraction protocol: this step, which employs you to wash the DNA pellet with 75% ethanol, was only preformed once.
 * <span style="font-family: Arial,Helvetica,sans-serif;">step 12 of the DNazol extraction protocol: pipetting of the DNA pellet was done almost violently with the 1mL micopipetter.
 * <span style="font-family: Arial,Helvetica,sans-serif;">step 1 of the DNA Quantification protocol: the program that runs the Nanodrop is called ND-1000, icon on the desktop.

<span style="font-family: Arial,Helvetica,sans-serif;">calculations <span style="font-family: Arial,Helvetica,sans-serif;">[RNA] = [concentration] x [A260] x Dilution Factor

<span style="font-family: Arial,Helvetica,sans-serif;">Results <span style="font-family: Arial,Helvetica,sans-serif;">Purified DNA target A260/280: 1.7 - 1.9 <span style="font-family: Arial,Helvetica,sans-serif;">Purified RNA target A260/280: 1.8 - 2.0 <span style="font-family: Arial,Helvetica,sans-serif;">Purified RNA target A260/230: 1.5 - 2.0

<span style="font-family: Arial,Helvetica,sans-serif;">Nanodrop results: <span style="font-family: Arial,Helvetica,sans-serif;">A260/280: 1.79 <span style="font-family: Arial,Helvetica,sans-serif;">A260/230: 0.27 <span style="font-family: Arial,Helvetica,sans-serif;">Abs: 67.7 <span style="font-family: Arial,Helvetica,sans-serif;">A-260: 18.2 <span style="font-family: Arial,Helvetica,sans-serif;">A-280: 10.15 <span style="font-family: Arial,Helvetica,sans-serif;">ng/ul: 910

<span style="font-family: Arial,Helvetica,sans-serif;">Conclusions <span style="font-family: Arial,Helvetica,sans-serif;">The resulting A260/280 of 1.79 for my DNA extraction was right on the money as far as what I was expecting. This was surprising, mainly because I man-handled the DNA pellet and skipped an ethanol washing (see notable deviations from procedure section). This result is interesting because it speaks to the robust nature of the protocol which is tolerant to mistakes.

<span style="font-family: Arial,Helvetica,sans-serif;">Reflections <span style="font-family: Arial,Helvetica,sans-serif;">The purpose of this lab was to introduce the methods of RNA and DNA extraction as tools for analyzing which genes are being expressed in organisms. Through DNA extraction and subsequent amplification, specific genes can be isolated. Through RNA extraction and the use of micro-arrays, expression levels of specific genes can be quantified. Both of these methods could be useful when investigating how environmental stressors impact an organism's tissues or metabolism. However, what was unclear today was how you select genes for isolation and how you design reagents to splice DNA into pieces for amplification. More background on the entire process of DNA isolation and amplification would have helped but the extraction in perspective.

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