Lidstrom:Gibson Assembly: Difference between revisions
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(New page: Design: - overlap by 20 bp Misc: - rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not ...) |
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Design: | Back to [[Lidstrom:Protocols]] | ||
== Janet's Tips == | |||
'''Janet is developing a protocol [[Janet B. Matsen:Guide to Gibson Assembly |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. | - Electrocompetence is ~ 1000x more effective so is tempting. | ||
- | - | ||
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. -
