EPF-Lausanne/27 October 2008

From 2008.igem.org

(Difference between revisions)

Latest revision as of 18:52, 29 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.

@@ Line 1: / Line 1: @@
-[https://2008.igem.org/wiki/index.php?title=EPF-Lausanne/20_October_2008 <<Previous] - [https://2008.igem.org/Team:EPF-Lausanne/Notebook Back to Notebook]
+[https://2008.igem.org/wiki/index.php?title=EPF-Lausanne/23_October_2008 <<Previous] - [https://2008.igem.org/Team:EPF-Lausanne/Notebook Back to Notebook]- [https://2008.igem.org/wiki/index.php?title=EPF-Lausanne/29_October_2008 Next>>]
+==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.