Team:Imperial College London/Protocol
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+ | <td style="background-color:#FFFF99;height:30px;width:150;text-align:center"><b>Enzymes used:</b></td> | ||
+ | <td style="background-color:#FFFF99;height:30px;width:150;text-align:center"><b>Required buffer:</b></td> | ||
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+ | <td style="background-color:#eeeeee;height:30px;width:150 px;text-align:center;">Prefix (vector)</td> | ||
+ | <td style="background-color:#eeeeee;height:30px;width:150 px;text-align:center;">SpeI & PstI</td> | ||
+ | <td style="background-color:#eeeeee;height:30px;width:150 px;text-align:center;">Buffer 2</td> | ||
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- | + | <td style="background-color:#eeeeee;height:30px;width:150 px;text-align:center;">Buffer 3</td> | |
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SpeI doesn’t cut particularly at the end of PCR products particularly well as there are few flanking bases. Can leave overnight and add the second enzyme as a second 90 minute cutting step. | SpeI doesn’t cut particularly at the end of PCR products particularly well as there are few flanking bases. Can leave overnight and add the second enzyme as a second 90 minute cutting step. | ||
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Revision as of 21:36, 27 October 2010
Lab protocols |
Listed below are the protocols we used for the project. We hope you find them useful! |
Lab Safety |
We have only worked with Category 1 organisms during the course of our project, and have not used toxic chemicals except when they are in appropriate solutions and therefore safe. All other lab safety measures were adhered to. |
Restriction Digests | ||||||||||||||||||||||||||||||||||
Method:
Reaction mixtures:
Or you can use this as a guide:
The buffer depends on the restriction enzymes used. Prefix Insertion:
SpeI doesn’t cut particularly at the end of PCR products particularly well as there are few flanking bases. Can leave overnight and add the second enzyme as a second 90 minute cutting step. |
Ligations |
A typical ligation reaction mixture is around 10 μl and contains
Insert mass (ng) = 6 x (Insert length (bp)/vector length (bp) x Vector mass (ng) Once the solution is made up, the tubes are vortexed and then spun down for around 10 seconds in a microcentrifuge. The ligation is done at 14°C in a water bath in the cold cabinet, and is left overnight. |
E. coli Transformations |
N.B. During pipetting the sides of the tube should not be touched to avoid contamination. Bubbles should be avoided because they can cause the cells stress.
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PCR |
PCR Reaction Mix
PCR programme
(-t°C optimal: 72°C for Taq // time optimal: 2-3kb/60 sec) (-t°C optimal:68°C for Pfu // time optimal: 1kb/15 sec)
We also used a positive control (other DNA to which the primers will definitely anneal) and a negative control (ddH20). The temperature cycle was as follows:
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Overnight Cultures |
Tubes containing 5ml of LB medium are inoculated with cells from one colony and then 5μl of antibiotic (for example chloramphenicol) is added. They are then left at 37°C overnight. |
SDS-PAGE | ||||
Short for: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
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