Week of 20211216 Troubleshooting of PCR After Gel Extraction, Mostly RPL11 (Nuclear Gene)

20211213 Re-PCR RPL11 and p47 Testing Diluting DNA 1:10 and Doubling DNA Input

  • Make diluted DNA:
Sample DNA Molcular grade water
3MLB-150 1 9
3mLB-UP 1 9
3mLB-LOW 1 9
  • Make RPL11 master mix on ice:
    • 1ul 10X NEB taq buffer * 8.8 = 8.8ul
    • 1ul 2mM dNTPs * 8.8 = 8.8ul
    • 1ul 25mM MgCl2 * 8.8 =8.8ul
    • 0.25ul 10uM vir_RPL11_F primer * 8.8 = 2.2ul
    • 0.25ul 10uM vir_RPL11_R primer * 8.8 = 2.2ul
    • 0.1ul NEB Taq * 8.8ul = 0.88ul
    • 0.4ul molecular grade water * 8.8 = 3.52ul
  • Added master mix, DNA, and water to strip tubes. Samples with “2” next to them had twice the amount of DNA added to them (~16-18ng total), and samples with the “D” were the 1:10 diluted samples
tube # sample ul RPL11 MM ul DNA ul water
1 3mLB-150-2 4 2 4
2 3mLB-UP-2 4 6 0
3 2mLB-LOW-2 4 4 2
4 3mLB-150-D 4 1 5
5 3mLB-UP-D 4 3 3
6 3mLB-LOW-D 4 2 4
7 neg control 4 0 6
8 pos control 4 1 5
  • Put in PCR program and geled the next day
  • Made p47 master mix on ice:
    • 1ul 10X NEB taq buffer * 8.8 = 8.8ul
    • 1ul 2mM dNTPs * 8.8 = 8.8ul
    • 1ul 25mM MgCl2 * 8.8 =8.8ul
    • 0.25ul 10uM p47_F primer * 8.8 = 2.2ul
    • 0.25ul 10uM p47_R primer * 8.8 = 2.2ul
    • 0.1ul NEB Taq * 8.8ul = 0.88ul
    • 0.4ul molecular grade water * 8.8 = 3.52ul
  • Added master mix, DNA, and water to strip tubes. Samples with “2” next to them had twice the amount of DNA added to them (~16-18ng total), and samples with the “D” were the 1:10 diluted samples (0.8-0.9ng)
tube # sample ul p47 MM ul DNA ul water
1 3mLB-150-2 4 2 4
2 3mLB-UP-2 4 6 0
3 2mLB-LOW-2 4 4 2
4 3mLB-150-D 4 1 5
5 3mLB-UP-D 4 3 3
6 3mLB-LOW-D 4 2 4
7 neg control 4 0 6
8 pos control 4 1 5
  • Put in PCR program and geled the next day
  • Next day a 1% gel was run for ~30 minutes at 90V
  • This gel came out really fuzzy and hard to read, it is not clear if there is primer dimer for RPL11 or actual amplification. I decided after this to go forward only troubleshooting the RPL11 amplification and also to run all my gels on 2% agarose to actually separate out something so small like primer dimer and a 137bp product

20211214 RPL11 Diluted PCR with 2% Gel

  • Tried doing the diluted DNA and then also doubling the amount of the diluted DNA
  • Made master mix on ice:
    • 1ul 10X NEB taq buffer * 8.8 = 8.8ul
    • 1ul 2mM dNTPs * 8.8 = 8.8ul
    • 1ul 25mM MgCl2 * 8.8 =8.8ul
    • 0.25ul 10uM vir_RPL11_F primer * 8.8 = 2.2ul
    • 0.25ul 10uM vir_RPL11_R primer * 8.8 = 2.2ul
    • 0.1ul NEB Taq * 8.8ul = 0.88ul
    • 0.4ul molecular grade water * 8.8 = 3.52ul
  • Added master mix, DNA, and water to strip tubes. Samples with “D-2” next to them had twice the amount of diluted DNA added to them (~1.6-1.8ng total), and samples with the “D” were the 1:10 diluted samples (0.8-0.9ng)
tube # sample ul RPL11 MM ul DNA ul water
1 3mLB-150-D 4 2 4
2 3mLB-UP-D 4 6 0
3 2mLB-LOW-D 4 4 2
4 3mLB-150-2 4 1 5
5 3mLB-UP-2 4 3 3
6 3mLB-LOW-2 4 2 4
7 neg control 4 0 6
8 pos control 4 1 5
  • Put in PCR program and geled the next day
  • Next day a 2% gel was run for ~30 minutes at 90V
  • Gel came out bad, could see the positive control but nothing else. I also poured the gel way too thick

20211215 Testing RPL11 PCR With Double Reaction Volume, GoTaq, and With Reduced PCR time

  • Wanted to try testing a 20ul PCR volume, GoTaq that Jessie said worked for her, and I wanted to modify the PCR times (reduce annealing and extension times) to see if they would work because the PCR program right now is very long
  • Because the diluted gel extracted DNA is the only thing that maybe worked, that’s what I used for the doubled reaction volume. I did a mix of sample types for the GoTaq, and samples that I knew worked for testing the PCR conditions
  • First I made the 2X reaction volume master mix:
    • 2ul NEB buffer * 7.7 = 15.4ul
    • 2ul dNTPs * 7.7 = 15.4ul
    • 2ul MgCl2 * 7.7 = 15.4
    • 0.5ul vir_RPL11_F primer * 7.7 = 3.85ul
    • 0.5ul vir_RPL11_R primer * 7.7 = 3.85ul
    • 0.2ul NEB Taq * 7.7 = 1.54ul
    • 0.8ul molec grade water * 7.7 = 6.16ul
  • Added master mix, DNA, and water to strip tubes. Samples with “D-2” next to them had twice the amount of diluted DNA added to them (~1.6-1.8ng total), and samples with the “D” were the 1:10 diluted samples (0.8-0.9ng)
  • I’m not sure where my calculations got a little off, I had to add a lot of water in per sample to make the reaction volume 20ul per sample
tube # sample ul RPL11 MM ul DNA ul water
1 3mLB-150-D-2 8 2 10
2 3mLB-UP-D-2 8 6 6
3 2mLB-LOW-D-2 8 4 8
4 3mLB-150-D 8 1 11
5 3mLB-LOW-D 8 2 12
6 neg control 8 0 12
7 pos control 8 1 11
  • Made master mix for GoTaq, 10ul reaction volume
    • 5ul GoTaq (2X) * 10 = 50ul
    • 4ul molecular grade water * 10 = 40ul
  • Added master mix, DNA, and water to strip tubes
tube # sample ul GoTaq MM ul DNA ul water
8 3mLB-150 9 1 0
9 3mLB-UP 9 1 0
10 3mLB-LOW 9 1 0
11 3mLB-150-D 9 1 0
12 3mLB-LOW-D 9 1 0
13 3mL HMW 9 1 0
14 2mL HMW 9 1 0
15 neg control 9 0 1
16 pos control 9 1 0
  • These were placed in the same PCR thermocycler with the regular PCR program
  • 95 degrees C 3 minutes
  • 95 degrees C 30 seconds
  • 57 degrees C 1 minute
  • 68 degrees C 1 minute 30 seconds
  • 68 degrees C 5 minutes
  • 12 degree C hold
  • bold sections are cycled 34 times
  • Made a separate PCR reaction set up for testing different PCR program times
  • Made master mix on ice:
    • 1ul 10X NEB taq buffer * 8.8 = 8.8ul
    • 1ul 2mM dNTPs * 8.8 = 8.8ul
    • 1ul 25mM MgCl2 * 8.8 =8.8ul
    • 0.25ul 10uM vir_RPL11_F primer * 8.8 = 2.2ul
    • 0.25ul 10uM vir_RPL11_R primer * 8.8 = 2.2ul
    • 0.1ul NEB Taq * 8.8ul = 0.88ul
    • 0.4ul molecular grade water * 8.8 = 3.52ul
  • Added master mix, DNA, and water to strip tubes
tube # sample ul RPL11 MM ul DNA ul water  
1 3mL 8 1 1  
2 2mL 8 1 1  
3 3mLB-150-D   8 1 1
4 3mLB-LOW-D 8 2 0  
6 neg control 8 0 2  
7 pos control 8 1 1  
  • Placed these in a slightly modified PCR:
    • 95 degrees C 3 minutes
    • 95 degrees C 30 seconds
    • 57 degrees C 30 seconds
    • 72 degrees C 1 minute
    • 72 degrees C 5 minutes
    • 12 degree C hold
    • bold sections are cycled 34 times
  • All of these were geled the next day on a 2% gel, poured low, and run for 30 minutes at 90V