Imperial College/3 September 2008

Wetlab

Transformation of B.subtilis

• 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 95^oC for 30 seconds
  • 30 cycles, 30 seconds 95^oC, 1 minute at 60/58/56^oC, 5 minutes at 72^oC
• A negative control was used at 56^oC 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 single colony PCR with Fw1 and Rv2 (not tested) and Fw1 and Rv1 (tested) under the following conditions:
• Using the primer, Fw1 and Rv2 -
  • 1 cycle 95^oC for 30 seconds,
  • 10 cycles, 30 seconds 95^oC, 1 minute at 52/50/48^oC, 5 minutes at 72^oC
  • 20 cycles, 30 seconds 95^oC, 1 minute at 61^oC, 5 minutes at 72^oC
  • 1 cycle, 1 minute at 72^oC.
• Using the primers, Fw1 and Rv1 -
  • 1 cycle 95^oC for 30 seconds,
  • 10 cycles, 30 seconds 95^oC, 1 minute at 48^oC, 5 minutes at 72^oC
  • 20 cycles, 30 seconds 95^oC, 1 minute at 61^oC, 5 minutes at 72^oC
  • 1 cycle, 1 minute at 72^oC.
• A negative control was also used where no primers were added.

Results

A 1% Agarose gel showing the results of various PCR reactions

• Lane 1 - Annealing step 60^oC using verification primers,
• Lane 2- Annealing step 58^oC using verification primers,
• Lane 3- Annealing step 56^oC using verification primers,
• Lane 4- Annealing step 56^oC using verification primers, no DNA negative control,

• Lane 5 - Annealing step 52^oC using AmyE fw1 and rev2 primers,
• Lane 6- Annealing step 50^oC using AmyE fw1 and rev2 primers,
• Lane 7- Annealing step 48^oC using AmyE fw1 and rev2 primers,
• Lane 8- Annealing step 61^oC using AmyE fw1 and rev1, positive control,
• Lane 9 - Annealing step 48^oC using AmyE fw1 and rev2, no DNA negative control,

• As we can see only for the positive control we see a band, this means that the verfication primers and the fw1 and rev 2 combinations are not working. We will double check the primer sequences and carry on trying to optimise the protocol.