Team:TU Delft/30 July 2010 content
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Revision as of 10:07, 2 August 2010
Alkane Sensing, Solvent Tolerance and Salt Tolerance
by Pieter
I tried to assemble the remaining biobricks from various sub projects. The goal is to assemble the following:
- AlkS + E0240
- PalkB + J06702
- J61101 + bbc1
- J61101 + PhPFDalpha
- J61101 + PhPFDbeta
To achieve this J06702, bbc1, PhPFDalpha and PhPFDbeta were amplified by PCR. The J61101 was cut open with S and P after which the biobricks were inserted. AlkS was inserted in the backbone with E0240.
PCR with PFX
Per sample the following mix was used:
- 1,5 ul 10 mM dNTPs
- 5 ul Enhancer
- 5 ul 10x PFX Buffer
- 1 ul 50 mM MgSO4
- 3 ul 5 pmol/ul Forward Primer
- 3 ul 5 pmol/ul Reverse Primer
- 0.6 ul PFX Polymerase
- 1 ul DNA Template
- 29.9 ul H2O
PCR Program: start:
- 2 min 95 C
- 1 min 50 C
- 1 min 68 C
repeated 25 times:
- 1 min 95 C
- 1 min 50 C
- 1 min 68 C
end:
- 10 min 68 C
PCR reactions
# | Description | Expected lenght (bp) | Primer | Status | Remarks |
1 | Marker | n/a | n/a | n/a | |
2 | J06702 | 869 | G00100 + G00101 | ✖ | |
3 | bbc1 | 703 | MF + MR2 | ✔ | |
4 | PhPFDalpha | 528 | MF + MR2 | ✔ | |
5 | PhPFDbeta | 430 | MF + MR2 | ✔ |