Lidstrom:Gibson Assembly: Difference between revisions
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Back to [[Lidstrom:Protocols]] | Back to [[Lidstrom:Protocols]] | ||
== Janet's Tips == | |||
'''Janet is developing a protocol [[Janet B. Matsen:Guide to Gibson Assembly |here]]''' | |||
==Design:== | ==Design:== | ||
| Line 15: | Line 18: | ||
*Electroporation can give much higher efficiency. For this reason, the assembled product is usually electroporated. | *Electroporation can give much higher efficiency. For this reason, the assembled product is usually electroporated. | ||
**rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells. | **rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells. | ||
** Janet | ** Janet electroporates 2 uL | ||
* | * | ||
Latest revision as of 13:21, 22 March 2013
Back to Lidstrom:Protocols
Janet's Tips
Janet is developing a protocol here
Design:
Fragments
- overlap by 40 bp
- efficiency
Oligo (primer) Design
PCR
Assembly
Transformation
- Electroporation can give much higher efficiency. For this reason, the assembled product is usually electroporated.
- rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells.
- Janet electroporates 2 uL
Screening
- Colony PCR 1st
- Sequence a few clones with primers that cover the "seams."
Misc:
- Electrocompetence is ~ 1000x more effective so is tempting. -
