Lab 6: tissue dissection, primer reconstitution, end-point PCR

Lab Objectives
  • Rehydrate, dilute primers so that they are ready to do PCR
  • Dissect tissues from your experimental animals (see last week's protocol)
  • Perform PCR on cDNA/gDNA with the primers you ordered


Primer reconstitution (to be explained in lab)
The primers you designed are in a dehydrated state. After re-hydrating them, we will be making 100uM stock and a 10uM working stock to use for PCR.

Polymerase chain reaction
Supplies and Equipment:
  • Micropipettes (1-1000 μl)
  • Sterile filter pipette tips (1-1000 μl)
  • Tip waste jar
  • PCR tubes (0.5 ml; thin walled)
  • 1.5 ml microcentrifuge tubes (RNAse free)
  • cDNA (student provided)
  • dNTPs
  • 2x GoTaq Green Master Mix
  • Primers
  • Nuclease Free water
  • thermal cycler
  • Kimwipes
  • microfuge tube racks
  • PCR tube racks
  • ice buckets
  • Lab coat
  • Safety glasses
  • Gloves


Procedure background
The polymerase chain reaction involves selective amplification of a DNA (genomic or complementary) target using the enzyme polymerase (after which the method is named), primers (short oligonucleotides), and dNTPs (A, C, T, and G). The method relies on thermal cycling, which consist of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. These heating and cooling cycles are comprised of three primary steps. A single cycle begins with denaturation at ~94°C and during this step the DNA is melted or rather unwound and the strands are pulled apart resulting in single stranded DNA. This is followed by an annealing step at ~50-60°C where the primers anneal to the target sequence. After which there is an extension step at ~72°C where nucleotides DNA polymerase synthesizes a new DNA strand by adding dNTPs that are complementary to the template. As PCR progresses, the newly generated DNA is also used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. This process allows us to generate thousands to millions of copies of a particular DNA sequence with only a single or a few copies of a piece of DNA.

For this lab we will be using Promega’s Go-Taq green master mix , please read the manufacture’s protocol.

PCR PROTOCOL

You will be preparing 50 µl PCR reactions: count how many total samples you have ( = x), add two negative controls (you will use nuclease free water instead of cDNA template), and add one extra reaction to account for pipetting error. For example, if I have 3 cDNA samples, I will calculate my master mix for 6 reactions.

1. Make a reaction mastermix in a 1.5 ml microcentrifuge tube labled "MM" and with your initials.


2.Prepare your mastermix by first calculating the total volume required for each component of the mastermix, then pipette each reagent and mix well. The following volumes are what you would need for a single reaction:

Reagent
1.reaction
x.reactions
5x GoTaq Green buffer
10 µL

10mM dNTP mix
1 µL

10 µM forward primer
1 µL

10 µM reverse primer
1 µL

GoTaq polymerase
0.25 µL

nuclease-free water
36.75




2. Pipette 48 ul of your master mix in to each of your 0.5 ml PCR tubes labeled with your sample name and with your initials

3. Add 2 ul of the appropriate template to each tube and mix via pipetting

4. Spin tubes to pool liquid at the bottom of the tubes. Load reactions into thermocycler making sure the caps are tightly secured.

5. Your samples will be put through the following thermal cycling profile and then stored at -20°C afterwards.


Step
Temperature
Time
Cycles
Denaturation
95C
5 min
1
Denaturation
95C
30 sec
40
Annealing
55C
30 sec
Extension
72C
90 sec
Final extension
72C
3 min
1
Hold
4C

1




Lab 6: protein SDS/PAGE and Western blot, analyze conventional PCR (via agarose gel) & qPCR data

Please print these lab materials before each lab.


Lab Objectives
  • Run extracted total protein from previous labs on SDS-PAGE Gel/Western Blot
  • Run conventional PCR samples on an agarose gel
  • Download qPCR data and discuss analysis

Making an agarose gel (This has been done ahead of time)

Supplies and Equipment:

  • Micropipettes (1-1000 μl)
  • Sterile filter pipette tips (1-1000 μl)
  • Tip waste jar
  • 1L flask
  • agarose
  • 1X TAE
  • Ethidium bromide
  • Microwave
  • Gel rigs
  • Kimwipes
  • Lab coat
  • Safety glasses
  • gloves

AGAROSE GEL POURING PROCEDURE
  1. Weigh 2g of agarose and mix with 150mL 1x TAE in a 1L flask
  2. 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. 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. Mix thoroughly by swirling, then pour into gel tray.
  5. Add gel combs. Using a clean pipet tip, pop any bubbles that could get in the way of your PCR product.
  6. After gel is set, wrap in plastic wrap (label with your initials and date) and place gel in the fridge if not using immediately.


Agarose Gel Electrophoresis
Last week you performed a conventional PCR in class using your reverse transcribed cDNA samples as template and primers your designed. This week we will be checking if amplification was successful using electrophoresis.


Procedure Background
  • Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.
  • The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20 ng DNA becomes distinctly visible. EtBr is a known mutagen, however, so safer alternatives are available.
  • A DNA ladder is a solution of DNA molecules of different lengths used in agarose gel electrophoresis. It is applied to an agarose gel as a reference to estimate the size of unknown DNA molecules
  • If amplification was successful you should see one clear band between 150-400bp (in length depending on your gene) and the negative controls will have no band. If there is a band in the negative control than there might be contamination in your reagents and you can not be sure that the intended gene was actually amplified.
  • Often contamination requires carful rePCR in case the contamination occurred during reaction setup. However, if any reagents are contaminated troubleshooting may be required to obtain a clean PCR product.
  • PCR bands outside of the intended size range could indicate unspecific amplification and will require either optimization of the reaction cocktail and thermal cycling parameters or redesigning the primers.


ELECTROPHORESIS PROCEDURE
  1. Place gel in gel box and fill with 1x TAE buffer (to fully cover wells)
  2. Remove combs from wells
  3. Load 7uL 100bp ladder in far left lane
  4. Load 20uL of your PCR sample into the gel (retain the remaining vol at -20ºC)
  5. Run gel at ~ 100V for ~ 1hr
  6. Visualize the gel on the UV transilluminator