Previously we have tried to test efficient integration into the B.subtilis genome. However, the primers we were using have so far failed to yield either a positive or negative result when a signle colony PCR was carried out on transformed B.subtilis. In order to check firstly if the primers are working and secondly if the single colony PCR protocol is working we carried out a series of testing. First to test the verification primers we are going to carry out a PCR on purified genome, that we have previously used successfully in a PCR reaction. We set up the following conditions:
1 cycle 95oC for 30 seconds
30 cycles, 30 seconds 95oC, 1 minute at 60/58/56oC, 5 minutes at 72oC
A negative control was used at 56oC where no genomic DNA was added.The results are shown in figure 1.1 below.
To confirm if the single colony PCR protocol is working we used this protocol with a new set of primers and a set that has been primers shown to work with purified genomic DNA. AmyE fw1 and rv1 (shown to work) are primers used to purify the 5' integration site from B.subtilis and AmyE fw2 and rv2 (not tested yet) are primers used to purify the 3'integration sites from B.subtilis. We performed the following reactions,
Results
A 1% Agarose gel showing the results of various PCR reactions. Each lane is loaded with 5ul of PCR reaction and 1ul of 6x sample buffer.
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