Lisa_441


 * 1/7/09 L1 - Tissue Extraction I **

Selected black abalone digestive gland tissue preserved in Liq N2 and stored @ -80C sample # 08:13-4 from 2nd (mini) WS resistance study TL: 61.0 mm TW: 31.7g SW: 12.16g Sample date: 8/14/08 Sump/Tank: Sump 1 (Exp)/2E (SNI) Notes: Active but not attaching, bloated – histo positive for weird (protist?) bug SEX: female Histo Score: FT atrophy: 0.5-1, PE RLO :  2.5, DG RLO: 1.5, Metaplasia: 0.5 Samples extracted in duplicate in case of error and also for replication ** RNA ISOLATION PROTOCOL ** *important to use extreme sterile technique when handling RNA 1. Turn on heating block to 55C. Also turn on spectrophotometer. 2. Add 500uL of TriReagent *caution: contains phenol* to a 1.5mL snap cap tube. Store on ice. 3. Using a clean razor blade, cut a piece of frozen tissue weighing between 50-100mg and add to tube containing TriReagent. // Tissue sample R1 = 63mg Tissue sample R2 = 50mg // 4. Carefully homogenize the tissue using a disposable pestle. 5. Add an additional 500uL of TriReagent to the tube and close the tube. 6. Vortex vigorously for 15s. //- Stop here for Lab 1 and freeze sample at -80 - Will continue extraction in Lab 2 // ** PROTEIN EXTRACTION PROTOCOL ** *important to use extreme sterile technique when handling proteases 1. Add 0.5mL of CelLytic MT solution to a 1.5mL snap cap tube. 2. Add 25mg of your tissue to the tube. Tissue sample P2 = 31mg // ** 3. Homogenize the tissue with a disposable pestle. 4. Close the tube and invert the tube several times. 5. Spin the tube in a refrigerated microfuge for 10mins. @ max speed. 6. While spinning, label a fresh tube with the word "Protein", source organism/tissue, your initials, and today's date. 7. Carefully transfer supernatant to labeled tube and store tube on ice. 8. To a fresh tube, add 1.5mL of Bradford reagent. Note: The Bradford assay works best when samples are mixed well. Invert tubes frequently during incubations, and immediately before measuring absorbance to ensure accurate absorbance readings.  *Also add a control tube (1.5ml Bradford and 30uL CelLytic MT solution) to “zero” out absorbance readings and account for any variations due to the CelLytic MT solution. 9. To this same tube, add 30uL of your protein extract. 10. Invert the tube several times and then incubate at RT for 10mins. 11. Mix the tube several times and transfer 1mL to a plastic, disposable cuvette. 12. Measure the absorbance at 595nm and record the value. **// P1 = 1.542 OD P2 = 1.571 OD //** (OD = optical density = absorbance) 13. Remove the cuvette from the spectrophotometer. Using a P1000 set to 1mL, carefully pipette the solution in the cuvette up and down a couple of times to mix. 14. Measure the absorbance at 595nm and record the value. **// P1 = 1.541 OD P2 = 1.568 OD //** 15. Repeat steps 13 and 14. Did not perform b/c values were almost identical 16. Average the three absorbance values you recorded. P2 Avg = 1.5695 OD //** // Note: Coomassie Blue dye in Bradford reagent absorbs at 595nm and the absorbance can be directly correlated to a specific amount of protein present in the sample when compared to a standard curve (already determined for us). // Standard curve was generated as per [|Manufacturers Instructions]. //Pierce: Coomassie (Bradford) Protein Assay Kit// 17. Plug your average absorbance that you just calculated into the following equation y=1011.9x to determine the concentration of protein in your sample. NOTE: It appears all of our protein values were outside of the curve. Need to dilute an aliquot of samples and run again. 18. Write the concentration on your tube <span style="color: rgb(0, 112, 192);">(forgot to do this) and place tube in TA ice bucket. Sample stored at -80C. // 4. //****// Design / order Primers to use in Lab 3 // ** **1/12/09 Ran a tblastn with all 4 genes in est_others [database] against Haliotis (taxid:6452) [organism]** code format="genbank" peptidoglycan recognition protein [Argopecten irradians] code [|AAR92030]
 * // 1. //****// Select tissue previously archived in ultra-cold freezer //**
 * // 2. //****// Start RNA extractions (add Tri-Reagent to samples and re-freeze) //**
 * // 3. //****// Extract protein from complementary sample //**
 * <span style="color: rgb(0, 0, 0);">// Tissue sample P1 = 32mg
 * // P1 Avg = 1.5415 OD

code format="genbank" peptidoglycan recognition protein S1 precursor [Chlamys farreri] code [|AAY53765]

code format="genbank" nuclear factor interleukin 3 regulated-like protein [Haliotis discus discus] code [|ACJ65688] code format="genbank" interleukin 17 [Crassostrea gigas]

code [|ABO93467]

EST match for PRG [|EB531075] (Evalue - 6e-21)

1/14/09 Last minute found something I think may be cool code format="genbank" lipopolysaccharide- and beta-1,3-glucan-binding protein [Chlamys farreri] code [|AAP82240]

EST match for LGBP [|CX726071] (Evalue 2e-80)

1/13/09 Primer3 Output (PRG)
code PRIMER PICKING RESULTS FOR gi|184186070|gb|EB531075.1|EB531075 aa344 Haliotis discus Hannailno Liver and Kidney cDNA library Haliotis discus hannai cDNA clone aa344 5', mRNA sequence

No mispriming library specified Using 1-based sequence positions OLIGO           [|start] [| len] [|     tm] [|    gc%] [|  any]  [|  3'] [|seq] LEFT PRIMER       176   20   59.84   60.00  3.00  0.00 GGTCACAGCCTCAACTCTCC RIGHT PRIMER      441   20   60.97   55.00  4.00  1.00 ACCCCCATCTTACCGATGAC SEQUENCE SIZE: 681 INCLUDED REGION SIZE: 681

PRODUCT SIZE: 266, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00

1 CTGGCAGTAAGGTAAAGTTTTGAAGGATCCTGCGTATATCTATCTTGTCATATATATTTT

61 CTGAAAATAAGCAAGGATGAAGACATTTTTCGGATGCCTACTGTTGGTATTAATGGCACC

121 ACGCTCTGAAGCGGCAGATTGCGCATGCGCCACATATGGCTTGCACGTGCGTTCCGGTCA >>>>>

181 CAGCCTCAACTCTCCTATCATCGGAACCATGACAAACGGTCAATGCGTGACCTTCAAGGG >>>>>>>>>>>>>>>

241 TGACCGGCAGGTGGCCGATGGCTACACCTGGGCGCACGTCGACTACAACGGAAAGGATGG

301 TTATGCCGCGGTCAACTGGCTCAACATTCATCCATGTGGGGCTCATAATAACGTTCTTCA

361 GCTGAGCGGCTGCCCGCACATCATCACACGTGCTGAATGGGGTGCACGCGCACCTAAATA

421 CGTCATCGGTAAGATGGGGGTCACTCCGAAGTATGTGTTCGTTCACCACGGTGCCACCGC <<<<<<<<<<<<<<<<<<<<

481 CCCATGTTATAACGAGGCTGCTTGCAAGGCCGAGGTCCTGTCTTATCAGAAGTATCACAT

541 GGACACGCACGGTTGGCCCGACATTGGCTACAGCTTCGTAGTCGGCGAAGACGGTCACGC

601 CTACGAAGCAAGAGGATGGGACACCATCGGCGCCCATACCTACGGCTACAACAGCGTCGG

661 ACTCGGTATCTGCGTTATTGG

KEYS (in order of precedence): >>>>>> left primer <<<<<< right primer

ADDITIONAL OLIGOS [|start] [| len] [|    tm] [|    gc%] [|  any]  [|  3'] [|seq]

1 LEFT PRIMER       176   20   59.84   60.00  3.00  0.00 GGTCACAGCCTCAACTCTCC RIGHT PRIMER      440   19   60.14   57.89  4.00  1.00 CCCCCATCTTACCGATGAC PRODUCT SIZE: 265, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00

2 LEFT PRIMER       425   19   60.14   52.63  3.00  1.00 ATCGGTAAGATGGGGGTCA RIGHT PRIMER      657   20   59.83   55.00  3.00  0.00 ACGCTGTTGTAGCCGTAGGT PRODUCT SIZE: 233, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

3 LEFT PRIMER       427   19   59.79   57.89  3.00  1.00 CGGTAAGATGGGGGTCACT RIGHT PRIMER      657   20   59.83   55.00  3.00  0.00 ACGCTGTTGTAGCCGTAGGT PRODUCT SIZE: 231, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

4 LEFT PRIMER       176   20   59.84   60.00  3.00  0.00 GGTCACAGCCTCAACTCTCC RIGHT PRIMER      308   19   60.28   52.63  2.00  0.00 CGGCATAACCATCCTTTCC PRODUCT SIZE: 133, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00 Statistics con  too    in    in          no    tm    tm  high  high        high sid many   tar  excl   bad    GC   too   too   any    3'  poly   end ered   Ns   get   reg   GC% clamp   low  high compl compl     X  stab    ok Left    5161     0     0     0   107     0  1127  3294   124    36     0    85   388 Right  5084     0     0     0     1     0   737  3785    11    16     0    97   437 Pair Stats: considered 1040, unacceptable product size 1023, high any compl 4, high end compl 2, ok 11 primer3 release 1.1.0

code Choose primer set 4 for PRG and submitted order

1/14/09 Primer3 Output (LGBP)
code PRIMER PICKING RESULTS FOR gi|82857336|gb|CX726071.1|CX726071 DGT151 Haliotis discus cDNA library (DGT) Haliotis discus cDNA, mRNA sequence

No mispriming library specified Using 1-based sequence positions OLIGO           [|start] [| len] [|     tm] [|    gc%] [|  any]  [|  3'] [|seq] LEFT PRIMER       119   20   59.56   55.00  2.00  0.00 GGCGTCCTCTACATCAAACC RIGHT PRIMER      354   20   59.53   45.00  4.00  0.00 TGAGCCGATATTTCCACCTT SEQUENCE SIZE: 632 INCLUDED REGION SIZE: 632

PRODUCT SIZE: 236, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00

1 CGACAATTTCGACACACTGGACTTCAAAGTGTGGGAACACGAGCTGACGGCTGGTGGGGG

61 AGGCAACTGGGAGTTTCAGTTCTACACCAACAACCGCACCAACACCTACGTCCGCGATGG >>

121 CGTCCTCTACATCAAACCCACGTTGACGGTGGACCAGTTTGGCGAGGCCTTCCTCACATC >>>>>>>>>>>>>>>>>>

181 CGGAAAACTTGAACTGTGGGGCGCAGGACCGCACGACACCTGTACCGGCAACGCCTTCTA

241 CGGCTGTGAGCGCGTCGGCAACCATCAGTACATCATCAACCCCATTCAGTCGGCCCGACT

301 CCGGAGCTCCAGGGGTCTGAACTTTAAATACGGCAAGGTGGAAATATCGGCTCAACTCCC <<<<<<<<<<<<<<<<<<<<

361 CAAGGGAGACTGGTTGTGGCCCGCCATATGGATGCTGCCGACGTACACGGAGTATGGCGG

421 TTGGCCGGCGTCCGGCGAGATTGACATCATGGAGAGCAGAGGTAACCGACACTACTACGA

481 CGCGAATGGCCGTTCTGTAGGAGTCGACTCTTACGGCAGTACCATTCACTTCGGCACCGA

541 CTACTTCCACAACGGCTGGTCACGTGCCCACCAGTCCTGGGTCAAAGAGAACGGAACTTA

601 CGGAGATGAGTTTCATACGTACGGAGTCGAGT

KEYS (in order of precedence): >>>>>> left primer <<<<<< right primer

ADDITIONAL OLIGOS [|start] [| len] [|    tm] [|    gc%] [|  any]  [|  3'] [|seq]

1 LEFT PRIMER       119   20   59.56   55.00  2.00  0.00 GGCGTCCTCTACATCAAACC RIGHT PRIMER      355   20   59.53   45.00  4.00  0.00 TTGAGCCGATATTTCCACCT PRODUCT SIZE: 237, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00

2 LEFT PRIMER       119   20   59.56   55.00  2.00  0.00 GGCGTCCTCTACATCAAACC RIGHT PRIMER      353   20   59.53   50.00  4.00  1.00 GAGCCGATATTTCCACCTTG PRODUCT SIZE: 235, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

3 LEFT PRIMER       336   20   59.53   45.00  4.00  0.00 AGGTGGAAATATCGGCTCAA RIGHT PRIMER      489   20   59.37   55.00  4.00  0.00 CCATTCGCGTCGTAGTAGTG PRODUCT SIZE: 154, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

4 LEFT PRIMER        23   20   60.13   45.00  4.00  0.00 TTCAAAGTGTGGGAACACGA RIGHT PRIMER      292   19   59.90   52.63  2.00  0.00 CGACTGAATGGGGTTGATG PRODUCT SIZE: 270, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00

Statistics con  too    in    in          no    tm    tm  high  high        high sid many   tar  excl   bad    GC   too   too   any    3'  poly   end ered   Ns   get   reg   GC% clamp   low  high compl compl     X  stab    ok Left    4653     0     0     0    28     0   680  3374   129    52     0    87   303 Right  4607     0     0     0    28     0   707  3289    17    10     0    93   463 Pair Stats: considered 662, unacceptable product size 578, high any compl 65, high end compl 3, ok 16 primer3 release 1.1.0

code Chose initial primer set for LGBP and submitted order

//**1. re-spec dilution of protein sample**//
//Set up 3 protein dilutions; 1:1, 1:3, 1:5 for both protein samples and re-spec'd P1-1:1 = 1.112 OD P1-1:3 = 0.729 OD 1:5 dilution within range of our standard curve **(y = 1011.9x)**
 * P1-1:5 = 0.570 OD**
 * P2-1:5 = 0.654 OD**//

//P1y = 576.78 P2y = 661.78 Back calculation from 1:5 dilution (6 parts total; multiply # by 6) P2 = 3,970.70ug/ml = 3.971ug/ul**//
 * P1 = 3,460.70ug/ml = 3.461ug/ul

**//2. Continue with RNA extraction protocol//**
//-Samples frozen at -80// //Turn on heating block to 55C.//

7. Incubate tube at room temperature (RT) for 5 mins. 8. 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. 9. Vortex vigorously for 30s. You are vortexing correctly if the solution becomes a milky emulsion. 10. Incubate tube at RT for 5 mins. 11. Spin tube in refrigerated microfuge for 15 mins. @ max speed. 12. Gently remove tube from microfuge. Be sure not to disturb the tube. 13. Slowly and carefully transfer most of the aqueous phase (the top, clear portion) to a fresh microfuge tube. **Do NOT transfer ANY of the interphase (the white, cell debris between the aqueous and organic phase).** 14. 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. 15. Add 500uL isopropanol to the new tube containing your RNA and close the tube. 16. 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". 17. Incubate at RT for 10 mins. **Incubated for 15mins** 18. Spin in refrigerated microfuge at max speed for 8 mins. 19. A small, white pellet (RNA and salts) should be present. If not, do not fret. Continue with procedure. 20. Remove supernatant. 21. 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. 22. Spin in refrigerated microfuge at 7500g for 5mins. 23. Carefully remove supernatant. Pellet may be very loose. Make sure not to remove pellet! 24. Briefly spin tube (~15s) to pool residual EtOH. 25. Using a small bore pipette tip (P20 or P200 tips), remove remaining EtOH. 26. Leave tube open and allow pellet to dry at RT for no more than 5mins. 27. Resuspend pellet in 100uL of 0.1%DEPC-H2O by pipetting up and down until pellet is dissolved. 28. Incubated tube at 55C for 5mins. to help solubilize RNA. 29. Remove tube from heat, flick a few times to mix and place sample on ice. This will be your stock RNA sample. 30. Quantitate RNA yield using spectrophotometer **(will do lab3)**

//**3. Run SDS-PAGE protein gel**//
1. //Begin boiling water on hot plate.// 2. Thaw you protein extract from last week. Mix well by inverting tube several times. 3. In a fresh, 1.5mL SCREW CAP tube add 15uL of your protein sample and 15uL of 2X Reducing Sample Buffer. 4. Mix sample by flicking. Briefly centrifuge (10s) to pool liquid in bottom of tube. 5. Boil sample for 5 mins. 6. While sample is boiling, observe assembly of gel box and gels. Rinse gel wells thoroughly as demonstrated. 7. When sample is finished boiling, immediately centrifuge for 1min. to pool liquid. 8. Slowly load your entire sample into the appropriate well using a gel loading tip. 9. Put lid on gel box and plug electrodes into appropriate receptacles on the power supply. 10. Turn power supply on and set voltage to 150V. Run for 45mins. 11. Add ~150mL (does not have to be measured - just need enough to cover the gel) of Coomassie Stain to a designated container. 11. Turn off power supply and disconnect gel box from power supply. 12. Remove lid from gel box. 13. Disengage the tension wedge. 14. Remove gel from gel box. 15. Carefully crack open cassette to expose gel. 16. Trim wells at top of gel. 17. 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) 18. Place gel into container with Coomassie Stain. 19. Incubate on shaker/rocker for 5 mins. 20. Carefully pour stain back into original container. Be careful not to dump out gel! 21. Rinse gel briefly with 10% acetic acid and pour this wash down the drain. 22. Add ~250mL (no need to measure) 10% acetic acid to container with gel. Incubate on shaker/rockers for 15mins. Change out buffer and repeat until bands become clearly visible. This may need to incubate O/N. If so, cover container with plastic wrap and leave on shaker/rocker.
 * PROTEIN GEL PROTOCOL** - See also [|Manufacturers Protocol / Manual: Precise™ Protein Gels]

1. ladder **(10uL)** 2. blank 3. Brianna 4. Brianna 5. blank 7. Lisa (30uL = 15uL protein + 15uL 2X Reducing Sample Buffer)** 8. Blank 9. Mac 10. Mac 11. blank 12. Ladder **(10uL)**
 * Lane ID**
 * 6. Lisa (30uL = 15uL protein + 15uL 2X Reducing Sample Buffer)
 * Figure 1: Brianna/Lisa/Mac FRONT;** **P1 sample [**//**3.461ug/ul**//**]** **in duplicate** - 15uL protein sample/well = **51.915ug/well**
 * **When cutting the corner on the FRONT GEL we accidentally cut it off at lane 12 instead of lane 1. So the FRONT GEL lane 1 is on the far right. Regardless, my samples are still the middle lanes.**
 * **My bands for both P1 and P2 look alot darker than Brianna (ab gill?) and Mac's (oyster gill?). Perhaps due to the dark green pigment of my sample since using digestive gland (possible inhibitors?). I do notice some dark banding representing Myosin and Phosphoraylase B.**

Figure 2: Brianna/Lisa/Mac BACK; P2 sample [//3.971ug/////ul//] in duplicate **- 15uL protein sample/well =** 59.565ug/well

[|Invitrogen Ladder]


 * 1/21/09 Lab 3 Reverse Transcription and PCR **

-Used nanodrop technology (sweetness!); calibrated machine with H2O, placed 1uL of RNA extract on platform. R2 = 1,603.0ng/ul **
 * //1.//****// Quantify RNA //**
 * R1 = 2,523.2ng/ul

REVERSE TRANSCRIPTION PROTOCOL 1. Mix your stock RNA sample by inverting tube several times. 2. Transfer 25ug of your RNA (.25ug of mRNA) to a fresh PCR tube. Bring the volume up to 5uL with PCR water. If necessary, spin tube briefly to pool liquid. -Add negative control here. -Because we had enough sample to spare and our [RNA] was within the 25ug range we just used 5ul of RNA (no water was added). //3. Incubate tube at 75C for 5mins in thermal cycler.// 4. Transfer tube IMMEDIATELY to ice and incubate for at least 5mins. 5. Make Master Mix 4 ul 5x Buffer (AMV RT Buffer) 8 ul dNTPs (10 mM total) 1 ul [|AMV RTranscriptase] 1 ul Oligo dT Primer 1 ul RNase free water Total = 15 ul -Made enough for 3 rxns but should have made extra, was short a few ul’s for the negative control Add 15ul MM to tube with diluted RNA in it (total volume now 20 ul) Vortex Spot spin Incubate at RT for 10 min Incubate at 37C for 1 hr in thermocycler Heat inactivate @ 95C for 3 min Spot spin Leave on ice or store at –20C **// 3. Perform PCR //** Run each template in duplicate AND make sure to include at least 2 negative controls for each primer (no template). //For a 50μl reaction volu//me: **Recipe** 25ul Taq 1ul Forward 1ul Reverse 21 H20 2ul Template Made enough MM for 9 rxns Load reactions into thermocycler. Overnight at 4C
 * // 2. Reverse Transcribe RNA to complementary DNA //**
 * Steps 3 & 4 were skipped by accident…OOPS!
 * PER RXN**
 * ** Component ** || ** Volume ** || ** Final Conc. ** ||
 * GoTaq®Green Master Mix, 2X || 25 || 1x ||
 * upstream primer, 10μM || 0.5–5.0μl || 0.1–1.0μM ||
 * downstream primer, 10μM || 0.5–5.0μl || 0.1–1.0μM ||
 * DNA template || 1–5μl || <250ng ||

** 1/22/09 ** Made 1.5% Agarose Gel Ran out PCR products on agarose gels and photographed -Used 100bp invitrogen ladder (7ul) -Added 15ul product/well -Ran 115V for 60min **TOP:** PRP primers (peptidoglycan recognition protein) Lane 1: 100bp ladder Lane 2: P1 Lane 3: P1 Lane 4: P2 Lane 5: P2 Lane 6: reverse transcription negative control Lane 7: reverse transcription negative control Lane 8: PCR negative control Lane 9: PCR negative control Lane 1: 100bp ladder Lane 2: L1 Lane 3: L1 Lane 4: L2 Lane 5: L2 Lane 6: reverse transcription negative control Lane 7: reverse transcription negative control Lane 8: PCR negative control Lane 9: PCR negative control Both thermocycler conditions should be optimized for each specific primer set.
 * Product coming up between 100 and 200bp but should be amplifying a 266bp product. Sequence was based off Atlantic bay scallop, may be different size in abalone. Negative controls look clean. Primer dimer issues. However, no dimer in my duplicate reverse transcription negative control. Weird!
 * Will excise and sequence my product.
 * BOTTOM:** LGBP primers (lipopolysaccharide- and beta-1,3-glucan-binding protein)
 * Super bright big band ~1,000-1,500bp. What could it be? genomic DNA carryover? (solutions: DNAse; design new primers that contain sequence half before and half after introns - check out homologs in sea urchin)
 * Faint band coming up between 200-300bp and my product is 236bp so that’s cool. Negative controls look clean. Still primer dimer issues. Might run gel again and excise/clean-up smaller band for sequencing.

1. //Run out protein samples as done previously (as in lab 2)//
Set up and run by Mac prior to lab

**2. //Transfer proteins from gel to nitrocellulose membrane//** **Western Blotting Protocol**
//General Guidelines//** • Avoid touching the working surface of the membrane, even with gloves. • Work quickly when changing solutions as membranes dry quickly. If the membrane dries, re-wet the membrane with methanol and rinse with water before proceeding. • Add solutions to the trays slowly, at the membrane edge, to avoid bubbles forming under the membrane. Decant from the same corner of the dish to ensure complete removal of previous solutions.
 * [|Western Breeze Manufacturer's Protocol]

1. Cool the transfer buffer to 4°C. 2. Soak the filter paper, membrane and gel in Transfer Buffer for 15 minutes. 3. Assemble the blotting sandwich in a semi-dry blotting apparatus (transfer cell- biorad) as follows: • Anode (+++) • 2 thick pieces of Filter paper - roll filter papers from middle to edge to remove bubbles and excess buffer. • Nitrocellulose Membrane -DO NOT TOUCH! • Gel - roll gel wet with buffer • Filter paper - roll again • Cathode (– – –) 4. Transfer the blot for 30 minutes at 20V. 5. Remove the gel from the sandwich and rinse with transfer buffer. 6. Use a cotton swab to remove any adhering gel from the membrane.

**3. //Probe membrane with antibody (HSP)//**
1. Prepare 20 mL of Blocking Solution Ultra filtered Water 14 ml Blocker/Diluent (Part A) 4 ml __Blocker/Diluent (Part B) 2 ml__ Total Volume 20 ml 2. Place the membrane in 10 ml of the appropriate Blocking Solution in a covered, plastic dish provided in the kit. Incubate for 30 minutes on a rotary shaker set at 1 revolution/sec. 3. Decant the Blocking Solution. 4. Rinse the membrane with 20 ml of water for 5 minutes, then decant. Repeat once. 5. Prepare 10 mL of Primary Antibody Solution (1:3000 dilution) Blocking Solution 10 ml __HSP 70 antibody 3.3 µl__ Total Volume 10 ml 6. Incubate the membrane with 10 ml of Primary Antibody Solution for OVERNIGHT

7. Prepare Antibody Wash Ultra filtered Water 150 ml __Antibody Wash Solution (16X) 10 ml__ Total Volume 160 ml 8. Decant Primary Antibody Solution (SAVE- can be re-used) 9. Wash the membrane for 5 minutes with 20 ml of prepared Antibody Wash, then decant. Repeat 3 times. 10. Incubate the membrane in 10 ml of Secondary Antibody Solution for 30 minutes, then decant. 11. Wash the membrane for 5 minutes with 20 ml of Antibody Wash, then decant. Repeat 3 times. 12. Rinse the membrane with 20 ml of water for 2 minutes, then decant. Repeat twice. 13. Incubate the membrane in 5 ml of Chromogenic Substrate until purple bands develop on the membrane. Development is complete in 1 to 60 minutes. 14. Take a digital picture of the membrane 15. Rinse the membrane with 20 ml of water for 2 minutes. Repeat twice 16. Dry the membrane on a clean piece of filter paper to open air
 * 1/29/09**
 * NEXT DAY**


 * We took 3 images of our gel: 5 minutes after development, 1.5 hours after development and the "overlay", which is a picture of the membrane overlaid with the coomassie gel (lined up using the markers).



2/4/09 Lab 5 QPCR using Syto-13

 * Syto-13 = non specific DNA binding dye fluoresces when bound to any double stranded DNA.
 * increase [DNA], increase binding sites, increase fluorescence.
 * Rxn specificity determined soley by primers

Each template run in duplicate AND 2 negative controls for each primer set.

1. Prepare master mix //For a 50μl reaction volu//me: Immomix - 25uL F - 2uL R - 2uL Syto - 2uL H20 - 17uL
 * **Component** || **Volume** || **Final Conc.** ||
 * Master Mix, 2X (Immomix) || 25µL || 1x ||
 * Syto-13 dye (50uM) || 2-5µL || 2 - 5µM ||
 * upstream primer, 10μM || 0.5–5.0μl || 0.1–1.0μM ||
 * downstream primer, 10μM || 0.5–5.0μl || 0.1–1.0μM ||
 * Ultra Pure Water || to 48uL || NA ||
 * RECIPE**

2. Add mastermix to wells of a white PCR plate 3. Thaw cDNA samples. 4. Add 2uL cDNA template to each reaction. 5. Add 2uL of ultra pure water to the negative control wells. 6. Cap the wells securely. 7. If necessary, spin the strips to collect volume in the bottom of the wells. 8. and ensure the lids are clean (wipe with a Kim Wipe) before going into the Opticon. 9. Load the plate, verify the PCR conditions and start the run.


 * Question of the Day**: How can you tell if you have gDNA carryover?
 * Answer:** Run a PCR on your RNA sample. If you get a product, you have gDNA carryover. RNA won't work on PCR bc it's unstable (will denature) and you use DNA polymerase in PCR.

**QPCR Results**
C1 N/A L neg C2 N/A L neg C3 30.2 L2 C4 33.01 L2 C5 26.12 L1 C6 25.26 L1 C7 N/A P neg C8 N/A P neg C9 27.07 P2 C10 19.64 P2 C11 18.85 P1 C12 19.62 P1
 * Row Ct Sample**
 * For both primer sets it looks like sample 2 has the greatest discrepancy among duplicate runs.


 * QPCR Melt curve (dissociation) analysis - amount of fluorescence vs temp - used to differentiate btwn specific anf non specific amplicons based on melting temp. My results show 2 peaks - this is bad news bc it means I am amplifying more than 1 product. Think about increasing annealing temp (increases specificity).


 * 2/11/09** [[file:507 project outline.doc]]


 * 2/12/09** Blk Ab Gill RNA extraction [[file:Blk Ab Resist project #2_GILL_RNAlater.xls]]


 * 2/13/09** Decided to switch sample set and work on Blk Ab Resistance Project #1 using DG samples stored in LN2 (still same project concept)

Google Doc - Abalone Exp1 DG
 * 2/18/09** Gathered ~80 DG samples and moved to Roberts Lab


 * 2/23/09** Extracted RNA from 5 samples using

[|RNA PowerSoil™ Total RNA Isolation Kit]
Sample 08:3-10 @ Step 8 had milky aqueous phase Step 11 - no visible pellets Step 12 - used 4mL of SR4 instead of 5mL
 * Note**:

- [|DNase] Treatment of 5 DG samples Step 1 Recipe - 1ul DNase, 5ul buffer, 44ul RNA Step 3 - 5ul DNase inact reagent
 * 2/24/09** RNA Quant on 5 DG samples via Nanodrop (data on google doc spreadsheet)


 * 2/27/09** [[file:Crosson_507ResearchProposal.doc]]

Recipe for 7 rxns 28ul buffer 56ul dntp 7ul AMV RT 7ul dT primer 7ul H2O total volume/cDNA sample = 20ul - Received primers for genes of interest (rab7, tollip, b-glucan, MnSD, PGRP, PGRP2, actin) - DNased 07:12 DG samples (n=19; missing 07-12-19) Step 1 Recipe - 1ul DNase, 1.5ul buffer, 12.5ul RNA Step 3 - 2ul DNase inact reagent total volume/ RNA sample after DNase = 10ul
 * 3/2/09** RT 5 DG samples [|AMV RTranscriptase] - used 5ul DNased RNA

88ul buffer 176ul dntp 22ul AMV RT 22ul dT primer 44ul H2O total volume/cDNA sample = 20ul
 * 3/3/09** RT 07:12 samples - used 4ul DNased RNA
 * Recipe** for 22 rxns

Recipe (per 25ul rxn) 12.5ul sensimix 0.8ul F 0.8ul R 0.5ul Sybr 9.4ul H20 Thermal Profile 95 - 7min 50 cycles of 95 - 10s 60 - 30s 72 - 30
 * 3/4/09** Sybr Green QPCR (Sensimix) testing all primer sets (n=7 from Roberts lab + n=1 of PGRP I designed) on 2 samples (06:5-31 SNI Exp Mort and 06:6-43 Carmel Exp Mort) - used only 1ul of template cDNA/rxn to preserve sample


 * After looking at the results I will use the following working primer sets to run the rest of my samples for the project: actin, tollip, rab7, and pgrp (lisa)
 * Other primer sets should be re-designed/optimized