2 September 2008

Wet Lab

Cloning

• Pfu PCR reactions for both AmyE integration sequences and the Laci gene were set up according to the standard [http://openwetware.org/index.php?title=IGEM:IMPERIAL/2008/Prototype/Wetlab/PCR protocol], using the optimal conditions determined yesterday and results run on a gel along with Aad9 Taq polymerase condiitons test (54^oC, 52^oC or 50^oC for the first 10 cycles then 60^oC for the last 20 cycles). Apropraite negativbe controls were used. For results, see tomorrow's entry.
• XL1-Blue cells containing the constructs in plasmids from GeneArt were grown up and incoulated into an overnight culture for midiprep tomorrow

Transformation of B.subtilis

• In order to confirm the transformations of B.subtilis that were performed in the previous weeks, we have designed validation primers. These validation primers are complementary to regions flanking the amyE locus and as a results the PCR product size will reflect any inserted DNA within this region. Using these primers a single colony PCR was performed, (link to prorotcol for single colony PCR for verficication.
• The results of this protocol are shown on figure 1 a 1% agarose gel. The lanes concerned are 1-4 and represent different annealing temperatures used, which were as follows:
  • 1-62 ^oC
  • 2-60 ^oC
  • 3-58 ^oC
  • 4-58 ^oC - Negative control - no DNA!!!
• If integration of the pDR110 plasmid was efficient we should see a band at 5.5kb, and if it has failed we should see a band at 2.5kb. We do not see these bands. The reason for this is unknown and so we will carry out further experiments, firstly to validate the verification primers and secondly to validate the signle colony PCR technique. To do this we have used primers previuously shown to work in a single colony PCR protocol and used the verfification primers with purified genomic DNA which has also previously been shown to work as a template. The method and results for this will be posted tomorrow.

Testing of Primers

• The following primers were tested today to confirm that they are working and if so the optimum annealing temperatures. The primers tested were the XylR and the EpsE 1 primers which were performed on purified genomic DNA. The protocol for this PCR reaction was following the standard protocol for PCR, click here
• The results of this protocol are shown on figure 1 a 1% agarose gel. The lanes concerned are 1-4 and represent different annealing temperatures used, which were as follows:
  • 5-51 ^oC - XylR
  • 6-49 ^oC - XylR
  • 7-47 ^oC - XylR
  • 8-46 ^oC - XylR
  • 9-46 ^oC - Negative control - no DNA!!!
  • 10-50 ^oC - AmyE 1 positive control as previously shown to work.
  • 11-53 ^oC - EspE1
  • 12-51 ^oC - EspE1
  • 13-49 ^oC - EspE1
  • 14-47 ^oC - EspE1
  • 15-47 ^oC - Negative control - no DNA!!!
  • 16-51 ^oC - AmyE 1 positive control as previously shown to work.
• Results - From the gel below it can be seen that the EpsE PCR reactions have worked, however with slight levels of contamination when a high annealing temperature is used. The XylR PCR reaction has not work efficiently, clearly visible from the faint bands. This means that the temperatures for annealing need to be optimised further.

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.