Team:Heidelberg/Templates/Indigoidine week8

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Revision as of 00:30, 5 October 2013


Indigoidine production with pMM-plasmids IV (Ilia)

  • run PCR for T and TE domain of bpsA for pMM64 validation and of svp for pMM65 validation:
File:PMM64-65 validation 170613.png
PCR for pMM64/65 validation (17-06-13); Ladder was missing, probably taken loading buffer by mistake; run at 100 V, 0.8 % gel (TAE); wanted amplicon size are: * bpsA_T: ~250 bp * bpsA_TE: ~850 bp * svp: ~800 bp
    • template each:
      • 5 ng of miniPrep (from 2013-06-15)
      • picked colony from transformed TOP10
      • water as control
    • polymerase: Taq (validation PCR)

used cycler protocol for svp and T domain of bpsA:

Cycles temperature [°C] Time [s]
1 95 180
12 95 30
71 (incr. down with 0.5 °C) 30
95 30
18 95 30
65 30
95 60
1 72 600
1 4 inf

used cycler protocol for TE domain of bpsA:

Cycles temperature [°C] Time [s]
1 95 180
12 95 30
63 (incr. down with 0.5 °C) 30
95 30
18 95 30
57 30
95 60
1 72 600
1 4 inf
  • retransformation from filter paper provided bei Fussenegger group
    • TOP10
    • with 10 µl pMM64 from original filter paper, plate on Amp, grow at 37°C
    • with 10 µl pMM65 from original filter paper, plate on Kan, grow at 37°C
  • prepare TB medium for ON (for comparement with LB)
  • colony PCR for plated pMM64 and pMM65 strains from 2013-06-16
  • prepare combinatorial experiment with gibson cloning or CPEC (polysytronic expression of bpsA and svp in TOP10)
  • prepare primer for bpsA only plasmid for BAP1 validation

Results and Discussion

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