October 1st, 2010
1. Make colony PCR with the transformations replated on September 29th:
- pSB1A2 + J23101 (SpeI-PstI restriction) + Δ RBS + GFP E0040: Colonies 1(tube 1, T1), 3 (T2), 5(T3) and 6 (T4), the later is expressing RFP.
- Religation of pSB3K3 + J23101 (SpeI restriction): 10 colonies (tubes 5-14)
- Religation of pSB4A5 (p30) + Minimmum Blue Promoter: Colonies 1-3 (T15-T17), 5 (T18), 7-10 (T19-T22).
- 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
- -60ºC 45 seg
- -72ºC 1.5 min
- 3. 72ºC 10 min
- 4. Hold 4ºC
2. Run gel to verify colony PCR.
Lanes: 1) Green ladder; 2) T2; 3) T1; 4-14) T3-T13; 15) Green ladder.
Lanes: 1) Green ladder; 2-10) T14-T22; 11) Green ladder.
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