Tripartite Conjugation in P. larvae

From 3-24-15.

Attempted PCR using the P11/P12 primers and thermocycler parameters described in the previous entry. This is the first time I have used these primers and am using a program that I designed myself. Amplicons were mixed with EZ vision loading dye using two different volumes of DNA since I had been having issues with overloading the wells in the past. Either 5 uL or 10 uL of amplicon DNA was combined with 2 uL of the loading dye and added to the wells of a 1% gel. The gel was ran for 40 minutes at 100 volts. Below is the resulting gel with labels below that:

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
Hi Lo	Transcon	pMarA	9545	Transcon	pMarA	9545	Empty

Lanes 2 - 4 were the 5 uL volumes of DNA and Lanes 5 - 7 were the 10 uL volumes.

Discussion:

The P11/P12 primers amplify the TnYLB-1 transposon. The "transcon" in Lanes 2 and 5 were a single transconjugate isolate that grew on the MYPGP Poly60 Kan100 plates after the tripartite conjugation. The P. larvae 9545 served as a negative control as it should not contain the transposon that is being amplified. pMarA contains the TnYLB-1 transposon and served as a positive control. The good news is that my thermocycler parameters are more than adequate at amplifying the TnYLB-1 transposon as there were bright and definite bands found in the positve pMarA control. The bad news is there doesn't appear to be a transposon in the transconjugate isolate. This transconjugate isolate tested positive using the AFB primers, indicating that it was P. larvae, however with these results today it means that the conjugation was not successful at inserting the transposon into it.

I may repeat this amplification test using the other transconjugate I have acquired in the future, however it is more likely I will re-attempt the tripartite conjugation and makes changes with the time intervals in hopes of improving my results.

//EWW