Tripartite Conjugation in P. larvae

From 1-7-15.
From 3-21-15.

I had previously ordered a couple sets of primers from Integrated DNA Technologies (IDT) on February 10th, 2015. These primers will be used to confirm that the TnYLB-1 transposon is indeed now present in P. larvae after tripartite conjugation. After the primers arrived I resuspended the primers using appropriate volumes of sterile ddH2O and stored them at -20C. I have designed thermocycler programs for each primer set based off a basic temp/time template to start with. The only variable that needed to be adjusted is the annealing temperature, which I have calculated to be within 5 degrees of both of the primers melting temperatures.

pMarA plasmid (from Le Brenton et al.)

Primers:

Primers P11 & P12 were created based on the primers from Xia et al. . These primers will amplify the internal fragment of the TnYLB-1 transposon that is found in the pMarA shuttle plasmid that I am using to transfer the transposon into P. larvae 9545.

P11 (Tm = 56.8C)
P12 (Tm = 56.4C)

Primer oIPCR1 & oIPCR2 were created based on the primers Le Brenton et al..This primer set amplifies outward from the TnYLB-1 transposon.

oIPCR1 (Tm = 45.8C)
oIPCR2 (Tm = 53.0C)

Thermocycler parameters:

Primer sets P11 & P12

	Temp	Time
Step 1	95.0C	2.00 m
Step 2	95.0C	1.00 m
Step 3	56.6C	1.00 m
Step 4	72.0C	1.00 m
Step 5	GoTo Step 2, repeat x 24
Step 6	72.0C	5.00 m
Step 7	20.0C	Forever

Primer sets oIPCR1 & oIPCR2

	Temp	Time
Step 1	95.0C	2.00 m
Step 2	95.0C	1.00 m
Step 3	49.5C	1.00 m
Step 4	72.0C	1.00 m
Step 5	GoTo Step 2, repeat x 24
Step 6	72.0C	5.00 m
Step 7	20.0C	Forever

//EWW