EPF-Lausanne/27 October 2008

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Revision as of 09:02, 29 October 2008

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Cloning

• Cloning of CD+ into AB+

First we tried to use AB+ as the insert, but then realized that AB+ and its vector both had almost equal sizes (around 2kb). It was therefore not possible to separate them using gel purification.

Fortunately, CD+ has 2.3kb, so we insert CD+ into AB+.

Again we use the modified ligation protocol:

• Reaction mix:
  • 3μl vector
  • 5.5μl insert
  • 1μl Ligase Buffer with ATP
  • 0.5μl T4 Ligase

Results:

There were thousands of transformants, and only around 20 transformants containing the autoligation of the vector.

However the control digestion gave us a result suggesting that the plasmid wasn't correct. There was a restriction cut at a size we didn't expect. We couln't solve this problem yet, but sequencing is on the way and will surely resolve the problem.

• Cloning of F2 into pHD1

We want to insert the part F2 (RBS-RhlI-Term) after the GFP found on the non-biobrick plasmid pHD1. To do this we need to do a blunt-end ligation. A restriction site for PciI (single-cutter) is found just after GFP.

Results:

We had only one transformant, but control digestion showed that it actually had inserted the right fragment.

We just don't know yet if it was inserted in the right direction.

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