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From 2010.igem.org

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