29 August 2008

WetLab

Midipreps

• Digested Midipreps of Biobricks - The midipreps of the following constructs were digested with EcoRI and SpeI in order to confirm the expected length of the biobrick. The following biobricks were midi preped, digested and analysed on agarose gel electrophoresis:
  • Terminator, size of biobrick = 129bp
  • AK3, size of biobrick = Should be no insert present
• The gel below shows the results of this analysis,

Lanes : M = Marker, 1 = Terminator Midi 1 Undigested, 2 = Terminator Midi 1 Digested, 3 = Terminator Midi 2 Undigested, 4 = Terminator Midi 2 Digested, 5 = AK3 Midi 1 Undigested, 6 = AK3 Midi 1 Digested, 7 = AK3 Midi 2 Undigested, 8 = AK3 Midi 2 Digested,

PCR

In a pursuit to verify the various PCR protocols and PCR primers we carried out the following reactions:

1. PCR with the pDR110 Vector using AmyE Fw1 and R1 primers
2. PCR with B.subtilis genomic DNA using AmyE Fw1 and R1 primers
3. Single Colony PCR using AmyE Fw1 and R1 primers and AmyE Fw1 and R2 primers

For each of these reactions a gradient of temperatures was used during the 10 cycles in the annealing stage of PCR and also the following 20 cycles, see our protocol for more information in summary the following reactions were carried out:

• 1. Vector - 10 cycles at 57^oC, 20 cycles at 64^oC, Primers = Fw1/R1
• 2. Vector - 10 cycles at 55^oC, 20 cycles at 62^oC, Primers = Fw1/R1
• 3. Vector - 10 cycles at 53^oC, 20 cycles at 60^oC, Primers = Fw1/R1
• 4. Genomic - 10 cycles at 57^oC, 20 cycles at 64^oC, Primers = Fw1/R1
• 5. Genomic - 10 cycles at 55^oC, 20 cycles at 62^oC, Primers = Fw1/R1
• 6. Genomic - 10 cycles at 53^oC, 20 cycles at 60^oC, Primers = Fw1/R1
• 7. Single Colony PCR - 10 cycles at 52^oC, 20 cycles at 64^oC, Primers = Fw1/R1, Sample of colony 1ul
• 8. Single Colony PCR - 10 cycles at 50^oC, 20 cycles at 64^oC, Primers = Fw1/R1, Sample of colony 1ul
• 9. Single Colony PCR - 10 cycles at 48^oC, 20 cycles at 64^oC, Primers = Fw1/R1, Sample of colony 1ul
• 10. Single Colony PCR - 10 cycles at 52^oC, 20 cycles at 64^oC, Primers = Fw1/R1,Sample of colony 2ul
• 11. Single Colony PCR - 10 cycles at 50^oC, 20 cycles at 64^oC, Primers = Fw1/R1,Sample of colony 2ul
• 12. Single Colony PCR - 10 cycles at 48^oC, 20 cycles at 64^oC, Primers = Fw1/R1, Sample of colony 2ul

Results

• Vector PCR - From the results the optimum conditions is clearly reaction 3 (10 cycles at 53^oC, 20 cycles at 60^oC, Primers = Fw1/R1)
• Genomic - From the results the optimum conditions is clearly reaction 5 (10 cycles at 55^oC, 20 cycles at 62^oC, Primers = Fw1/R1)
• Single Colony PCR - From the results clearly it is sufficient to use only 1ul of sample. In addition only the combination of Fw1 and Rv1 is working at either 10 cycles at 52^oC, 20 cycles at 64^oC or 10 cycles at 50^oC, 20 cycles at 64^oC