PCR

Date: 10/22/18

Rationale: Since plaque assays have been consistently turning up negative, I am trying a different methodology to find phage.

Procedure:

1. Spun down lysate at 3000g for 5 minutes.
2. Placed 1mL of lysate into micro-centrifuge tube.
3. Boiled sample to release phage DNA from capsid.
4. Made three PCR mix tubes and labeled each with a circle and a number correlating to each primer mix.
5. Each tube was filled with 12.5μL of Taq polymerase and dNTPs. Added 4μL of primer mix 1 to tube 1, primer mix 2 to tube 2, and primer mix 3 to tube 3. Added 1μL of lysate and 1μL of partners lysate. Filled up rest of tube with 6.5μL of DI water so that volume in tube is 25μL.
6. Labeled another three tubes with 12.5μL of Taq polymerase and dNTPs with a plus and a number correlating to each primer mix for positive controls.
7. Filled each tube with 4μL of primer mix corresponding to its number. Added 1μL of positive control lysate. Filled each tube up with 7.5μL to reach 25μL.
8. Gave the tubes to the TA to be run in the thermocycler.

Observations: When 1μL of lysate was added it didn’t mix with the rest of the solution and just stayed at the top of the tube. This is also the first time we are trying the clear Taq polymerase solution.

Conclusions and Next Steps: The amount of lysate could most definitely be increased. Usually the Taq polymerase used comes from a stock that has the dye already added. This could lead to no DNA being replicated by the Taq, as it might not work. Next class I will conduct a gel electrophoresis to see if I did in fact find phage DNA in my sample.