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Team:Heidelberg/Templates/Indigoidine week8
From 2013.igem.org
Indigoidine production with pMM-plasmids IV (Ilia)
- inoculate 5 ml LB + Kan with TOP10-pMM65
- miniPrep of pMM65 -> 23 ng / µl
- prepare TOP10-pMM65 glycerol stock
- 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)
- template each:
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
