Re-trying PCR

Last week, I tried Chelex extractions, gel, and PCR. However, my PCR blank was contaminated! Based on Carolyn’s advice, I redid my PCR using new GoTaq, primer, and NF water aliquots.

Methods

PCR:

1. Use RNAse AWAY to clean bench space, pipets, tip boxes, etc.
2. Label either an 8-strip PCR tube or a 96-well plate with sample ID and “P” (ex. 3P) and a 1.5 mL eppendorf tube “PCR MM.” Be sure to label an extra tube for the PCR blank.
3. Calculate the amount of reagents needed for PCR master mix for samples, a PCR blank, and two extra reactions.
   • GoTaq: 12.5 µL/sample
   • SMC F primer: 2.5 µL/sample
   • SMC R primer: 2.5 µL/sample
   • DEPC H₂O: 5.5 µL/sample
4. Preheat the thermocycler with the TD65_48L PCR protocol
5. Get ice and thaw PCR reagents and DNA at room temperature. Briefly vortex, spin down, and move onto ice as soon as reagents thaw.
6. Make new SMC F and R primer aliquots. Take the 100 µM stock bottles (blue lids) and dilute into a new, labelled 1.5 mL eppendorf tube in a 1:10 dilution (ex. 10 µL primer stock, 90 µL NF water).
7. Make master mix based on calculations in step 3 in the labelled PCR MM tube. Vortex, spin down, and keep on ice.
8. Aliquot 23 µL of PCR MM from step 6 in each tube for the samples, extraction blank, and PCR blank.
9. Once the DNA thaws, briefly vortex DNA and spin down. Place on an ice block tube holder.
10. Add 2 µL of DNA into each sample tube. Add 2 µL DEPC water for the PCR blank.
11. Seal wells completely. Use tube/well caps, foil plate seal, or Microseal A film to seal plates. Use roller to push down seal and use the plate sealer tool to ensure all wells are completely sealed.
12. Vortex the sealed tubes and briefly spin down.
13. Place in the thermocycler, close lid, and run TD65_48L PCR protocol.
14. When protocol is complete, place PCR product in the 4ºC fridge until ready to load a gel.

Gel:

1. Obtain PCR product and TriTrak from the 4ºC fridge and move into gel room.
2. Microwave pre-made gel mix in the microwave for 3 minutes in 1.5 minute intervals. Be sure to swirl the bottle to mix the gel liquid.
3. Allow the bottle with gel mix to cool for a few minutes. While cooling, tape off sides of the gel tray.
4. After bottle has cooled enough to the touch but the gel mix is still in liquid form, pour the mix into the taped gel tray slowly to avoid bubbles. Use combs to make wells by slowly inserting them into the gel to avoid bubbles.
5. Allow gel to harden.
6. Once gel is hardened, place in gel box with 1x TAE buffer.
7. Obtain a piece of parafilm and pipet 1 µL of TriTrak dye for each sample, extraction blank, and PCR blank. On the parafilm, the dye will bead up. Place the dots far enough apart to avoid contamination.
8. Take 6 µL of PCR product and mix with a 1 µL dot of TriTrak dye by pipetting up and down at least 10 times. Then, pipet up 6 µL and load gel. Repeat for each sample until halfway through samples. Then, add 6 µL of ladder + dye mix, and continue with remaining samples.
9. Run gel for 30 minutes.
10. After running gel, remove gel tray and image.

Results

Figure 1. Gel image for samples 3, 5, 10, 31, 32, 52, and 60 after re-trying PCR with clean reagents.

Good news: my PCR blank doesn’t have a band, so it wasn’t contaminated! Bad news: my extraction blank has a band so it’s contaminated. My suspicion is that the DEPC water above the molecular bench may be contaminated, since I used that for my extractions and initial PCR run. I’ll re-do the extractions, but using NF water from the freezer. Carolyn also suggested more thorough cleaning with RNAse AWAY between each tissue extraction and replacing the kim wipe I use for blotting in case any tissue “flakes” away while I’m removing ethanol from the tissue.

Going forward

1. Redo extractions with these samples to see if that fixes contamination issue
2. Continue with Chelex extractions, PCRs, and gels
3. Treatment-wise TTR analysis
4. Treatment-wise respirometry analysis
5. Get existing genotype data from Julia
6. Incorporate genotype into TTR and respirometry analyses
7. Prepare talk for PICES
8. Update methods and results of 2023 paper