the Transposition of Your Gene of Interest into the Viral Genome.
Prepare selective agar plates as in the electroporation and transposition
the appropriate number of 0.5 ml microcentrifuges tubes. Place on ice.
each tube add:
5 µl of 10X PCR buffer
1 µl of 10 mM dNTP mix
1.5 µl of 50 mM MgCl2
2.5 µl of primer mix (1.25 each 10 µM stock)
0.5 µl Taq Polymerase (2.5 U)
Add DDW to a final volume of 50 µl
the contents of the tube by gently tapping the tube.
a colony from the plate into the reaction mix.
PCR as follows:
a) 93oC for 3 min
b) 94oC for 45 sec
c) 55oC for 45 sec
d) 72oc for 5 min
perform steps b-d x40 cycles.
0.7% agarose gel in 1xTAE containing 0.5 µg/ml ethidium bromide
24µl of each sample on gel (prepare the sample as follows: 20 µl
of PCR reaction + 4 µl of 6x loading buffer).
Run gel at 100 mA.
expected results from the PCR are as follows:
Bacmid alone ~300 bp
Bacmid transposed with pFastBac1 ~2300 bp
Insertion of your target gene will result in increasing in the size of
the PCR product.
This increase corresponds to the size of your gene of interest.
(per liter) 242 g Tris base
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA pH 8.0
buffer: 0.25% bromophenol blue
0.25% xylene cyanol FF
40% (w/v) sucrose in water
Store at 4oC