August 9th, 2010
1. Plasmid extraction to BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040) strain incubated on August 8th.
- Plasmid extraction was done using the High Pure Plasmid Isolation Kit Roche.
2. Make PCR to change RBS into BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040), this because we have one construction with this plasmid + Blue Promoter + GFP E0240, and we want to have both GFP’s under the same RBS, so we can characterize better the promoters without noise caused if we used different Ribosome Binding Sites.
- PCR will be done with Taq Polymerase.
- I will use primers RBS_GFP FWD-J23101 REV
- PCR product is expected to be 3670 bp (941 biopart + 2729 primer pSB3K3).
- Tubes are marked 1-2:
- 1. BBa_I20260 RBS-GFP FWD-J23101 REV
- 2. Positive control: BBa_K137019 (~2855 bp) using primers Preffix FWD-Suffix REV.
- PCR with Taq DNA polymerase
- Reactive (ul x sample)
- Taq Polymerase -> 1
- Taq Reaction Buffer 10X -> 5
- MgCl 50mM (can be used up to 3ul) -> 2.5
- dNTP’s 0.4ug/ul -> 2.5
- Primer Forward (can be used up to 3ul) -> 2.5
- Primer Reverse (can be used up to 3ul) -> 2.5
- HPLC -> 32
- DNA -> 2
- Total volume -> 50
- Thermocycler program:
- 1. 95ºC 5 min
- 2. 35 cycles
- 95ºC 45 seg
- 60ºC 45 seg
- 72ºC 1.5 min
- 3. 72ºC 5 min
- 4. Hold 4ºC