Lab 3: Nested PCR
Procedure:
1.
We will again set up 6 PCR reactions for the second stage of PCR. Three will be experimental reactions, consisting of gDNA from your two plants, purple deadnettle, and from Arabidopsis, and two will be controls set up with pGAP plasmid DNA as a positive control and sterile water as a negative control.
Make a list of how you will label each nested PCR reaction below, including which template DNA will be in each tube.
2.
Prepare a master mix. Each nested PCR reaction volume is 40 uL, so each reaction requires 20 uL of master mix and yellow nested primers. Calculate how much master mix is required:
Volume of master mix required = (# reactions +1) x 20 uL = uL
The nested PCR uses primers that specifically target DNA within the fragments that were amplified in the last round of PCR. The yellow nested primers are supplied at a concentration of 25 uM, and we want our working concentration of primers to be 0.5 uM. Calculate the volume of nested primers required in the master mix using the formula M1V1=M2V2:
Required volume of nested primers (V2) =
3.
Label a microcentrifuge tube “master mix.” Add the required volume of master mix to the tube, and then add your primers to it and mix by pipetting up and down. Briefly spin it down in the microcentrifuge, and keep it on ice.
4.
Obtain tubes from the initial round of PCR. Use only the tubes that contained the gDNA, including the positive control gDNA, but not the plasmid DNA or sterile water reactions.
5.
Prepare the template DNA. Treat it with exonuclease I to remove excess primers from the initial PCR tubes. Using a fresh pipet tip each time, pipet 1 uL of exonuclease I into each initial PCR sample of amplified gDNA. Mix well by pipetting up and down.
6.
Incubate at 37 C for 15 minutes to allow the exonuclease to degrade the primers.
7.
Incubate at 80 C for 15 minutes to denature the exonuclease and stop the reaction.
8.
Label a microcentrifuge tube for each exonuclease-treated initial PCR tube.
9.
The initial PCR sample will be diluted 100 times in the second PCR. Rather than pipetting 0.4 uL of the initial PCR sample (1/100 of the 40 uL reaction volume), the sample is diluted in advance to allow a larger volume to be pipetted, and to reduce pipetting errors. To dilute each intial PCR sample to 1/50 the original concentration, pipet 98 uL of sterile water into each of the labeled microcentrifuge tubes.
Remember that the initial PCR sample will be diluted further when added to the master mix.
10.
Using a fresh pipet tip each time, pipet 2 uL of the appropriate initial PCR into the appropriate microcentrifuge tube. Close the cap.
11.
Flick the tube with your finger to mix (you may also vortex it briefly). Spin briefly in a microcentrifuge.
12.
Label PCR tubes according to the designations you made at the start of the lab, and place them in PCR tube adaptors on ice.
13.
Each PCR needs the following reagents, for a total volume of 40 uL:
Master mix with primers: 20 uL

DNA template or negative control: 20 uL
14.
Pipet 20 uL of your master mix into each PCR tube.
15.
Using a fresh pipet tip each time, add 20 uL of the appropriate DNA template (diluted gDNA, plasmid
DNA, or sterile water) to each PCR tube. Gently pipet up and down to mix reagents. Recap the tubes.
16.
Spin down the PCR tubes in their adaptors in the microcentrifuge briefly.
17.
Place tubes in the thermal cycler. We will use the following PCR program:
Stage 1: 95 C for 5 minutes (initial heating and denaturation)
Stage 2: 40 cycles of
Step 1: 95 C for 1 minute (denaturation)
Step 2: 46 C for 1 minute (annealing)
Step 3: 72 C for 2 minutes (extension)
Stage 3: 72 C for 6 minutes (final extension)
Hold at 15 C
18.
Store PCR products at -20 C.
19.
We will now pour agarose gels so that next week we can analyze our PCR products by electrophoresis.
You will share a gel with a partner, so pour only one gel for the two of you. We will mix up 1% agarose gels, so you will add 0.4 g of agarose to 40 mL of 1x TAE buffer in a 500 mL flask. Cover the flask with a paper towel and microwave it for 30 seconds, and then remove and swirl it. WEAR AUTOCLAVE
GLOVES!! Microwave for additional 10 second increments until agarose is fully dissolved. Allow the solution to cool for about 10 minutes (you should be able to touch the flask) and then put on latex gloves and add 2 uL of ethidium bromide and swirl. Ethidium bromide is a mutagen, so be careful not to let it touch any surfaces in the lab, and to avoid touching anything once you have handled it, until you remove your gloves. Any pipet tips or gloves that have touched EtBr should be discarded in the hazardous solid waste container in the lab. Allow the agarose solution to cool for an additional 15 minutes. In the meantime, set up your gel apparatus.
20.
Once your gel has cooled, pour it into the chamber and allow it to solidify for another 15 minutes.
When it has set up, it will look bluish and cloudy. Remove the combs and tape, and put your gel in a bag with your name on it. We will store these in the refrigerator until next week.
Further questions:
A.
Why did we conduct 2 rounds of PCR in this lab (both initial and nested PCR)?
B.
What would happen if we failed to denature the exonuclease after the digestion step in this protocol?
C.
Why do you think we can use a lower annealing temperature in the nested PCR program than in the initial PCR program? (Think about the rules of PCR and primer design we discussed in lecture)