Haynes Lab:Notebook/TB Biosensor/2012/06/08

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PCR'd the plasmids cut on 12.05.29 to PCR out the individual genes with the correct overhangs (Silver Standard RFC[23] compatible with no frame shift).

Plasmids from 12.05.29:

Lane Part Number Gene(s)
1 K118014 RBS + crtE
2 K274100 RBS + crtE + RBS + crtI + RBS + crtB
3 K274200 RBS + crtE + RBS + crtB + RBS + crtI +RBS + crtY
4 K118002 crtB
5 K118006 RBS + crtB
6 I742161 RBS + crtI + RBS + crtB
7 K118003 crtI
8 K118005 RBS + crtI


Lane Gene Plasmid Primer F Primer R
1 crtE 1 P001 P002
2 crtE 2 P001 P002
3 crtE 3 P001 P002
4 crtB 2 P003 P004
5 crtB 3 P003 P004
6 crtB 4 P003 P004
7 crtB 5 P003 P004
8 crtI 2 P005 P006
9 crtI 3 P005 P006
10 crtI 7 P005 P006
11 crtI 8 P005 P006


PCR reaction

Reagent Volume
DNA (plasmid) 1.0
10 μM primers 1.0
2x GoTaq 25.0
dH2O 23.0

--> Wang Lab PCR machine, block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec; 50°C/ 30 sec; 72 °C/ 1 min.] x30
  • 72 °C/ 3 min.
  • 4 °C/ ∞

> Qiagen PCR cleanup kit; elute w/ 50ul EB2O


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