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EPF-Lausanne/27 October 2008
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There were thousands of transformants, and only around 20 transformants containing the autoligation of the vector. | There were thousands of transformants, and only around 20 transformants containing the autoligation of the vector. | ||
Revision as of 20:06, 28 October 2008
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.
