Team:TU Delft/Notebook/cloning-plan
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==Two-way ligations strategies== | ==Two-way ligations strategies== | ||
+ | By using two-way ligaiton we ligate two parts to each other. The backbone of the smallest part was cut open. The largest part was cut out of its backbone. To prevent self-ligation we cut the backbone with an additional restriction enzyme. | ||
+ | |||
+ | In the case that the smallest part was the second part we used cloning strategy A | ||
+ | |||
+ | The smallest part was cut with EcoRI and XbaI | ||
+ | |||
+ | The largest part was cut with EcoRI and SpeI | ||
+ | |||
+ | It is important that the extra enzyme only cuts in the backbone and not in the part itself | ||
+ | |||
[[Image:TU Delft 2 way ligation A.tif]] | [[Image:TU Delft 2 way ligation A.tif]] | ||
+ | |||
+ | In the case that the smallest part was the first part we used cloning strategy B | ||
+ | |||
+ | The smallest part was cut with SpeI and PstI | ||
+ | |||
+ | The largest part was cut with XbaI, PstI | ||
+ | |||
+ | It is important that the extra enzyme only cuts in the backbone and not in the part itself | ||
[[Image:TU Delft 2 way ligation B.png]] | [[Image:TU Delft 2 way ligation B.png]] | ||
+ | |||
+ | In the end all BioBricks have to be cloned in the pSB1C3 plasmid backbone. With our strategy all the BioBricks are still in its original plasmid backbone, which in most cases was pSB1A2. We performed a pSB1C3 migration which is illustrated by cloning strategy C. | ||
Revision as of 06:48, 18 October 2010
Contents |
Cloning Plan
Once we decided what we wanted to achieve and with which genes, we designed the following cloning plan.
Two-way ligations strategies
By using two-way ligaiton we ligate two parts to each other. The backbone of the smallest part was cut open. The largest part was cut out of its backbone. To prevent self-ligation we cut the backbone with an additional restriction enzyme.
In the case that the smallest part was the second part we used cloning strategy A
The smallest part was cut with EcoRI and XbaI
The largest part was cut with EcoRI and SpeI
It is important that the extra enzyme only cuts in the backbone and not in the part itself
In the case that the smallest part was the first part we used cloning strategy B
The smallest part was cut with SpeI and PstI
The largest part was cut with XbaI, PstI
It is important that the extra enzyme only cuts in the backbone and not in the part itself
In the end all BioBricks have to be cloned in the pSB1C3 plasmid backbone. With our strategy all the BioBricks are still in its original plasmid backbone, which in most cases was pSB1A2. We performed a pSB1C3 migration which is illustrated by cloning strategy C.