Touchdown PCR, Nanodroping and Gels

 

After our successful PCR last week, we decided to start this week with another round of PCR in order to stockpile samples in case we runout again. 

We started by nanodroping our samples. Our left fragment measured in at 402.1 ng of DNA per microliter, our tet at 463.3 ng of DNA per microliter and our right at 503.0 ng of DNA per microliter.



After nanodroping the samples we diluted a small amount in order to reach 1ng/μl of DNA. We mixed one microliter of the left, tet and right fragments with 4μl, 4.5μl and 5μl of PCR water respectively. After diluting our sample, we moved on to assembling the PCR. We decided to make three 50 μl batches each for the left, tet and right fragments. Instead of doing our normal PCR, we decided to do touchdown PCR. 


PCR Tubes-

    2.5 μl forward primer

     2.5 μl reverse primer

    21.5 μl mastermix

    1 μl DNA sample

    22.5 μl PCR water


We ran out of master mix for the third tube of the right segment buy roughly 3 microliters. We decided to keep the tube and run it with the others anyways, despite us not expecting any results. 

After assembling the tubes we moved on to programing the PCR machine for touchdown PCR. We ran everything like normal, but lowered the annealing temperature by half a degree every cycle. 

    95°C for 4:00
    95°C for 0:30
    60°C for 0:30 (lowered .5°C every cycle)
    72°C for 0:30
        repeated 30 times 
    72°C for 7:00
        then held at 4°C 




After running the PCR we ran our samples on a 1.2% TAE 120 ml gel. We started at 80 milliamps, then moved to 100 after roughly 25 minutes. 

Our gel was laid out as such- 


However, after running our gel we only saw the two ladders, and the 2c, 3a, and 3b fragments. 




If we had seen no bands at all we would have decided it was something wrong with our process and mix. However as we have one tet and two right that have clearly come through on the gel, were not sure what went wrong. it may have been an issue with the diluting or the samples themselves. On monday we will renanodrop the original samples, but we do not intent to PCR with the intent of a stockpile again. 









Comments

  1. EricaOctober 13, 2022 at 5:26 PM

    Nice blog this week. Better luck in the lab next time.

    ReplyDelete

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