September 25th, 2010
1. Make colony PCR of the ligations:
- pSB3K3-J23101 + Δ RBS-GFP E0040 (3 colonies, tubes 1-3)
- pSB3K3-Min. Blue Promoter + Δ RBS-GFP E0040 (1 colony, tube 4)
Colony PCR methods:
- Take a colony and resuspend in 200ul of Tri-EDTA 10/1-NaCl 10 mM.
- Heat 10 min at 95ºC.
- Centrifugue at 14000 rpm 2 min.
- Take 10 ul as template for PCR.
- Primers used were Preffix FWD and Suffix REV, reactives needed for one reactions are as follows:
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 -> 24
- DNA -> 10
- Total volume -> 50
- 1. 95ºC 5 min
- 2. 35 cycles
- 95ºC 45 seg
- 55ºC 45 seg
- 72ºC 4 min
- 3. 72ºC 10 min
- 4. Hold 4ºC