Melbourne/Site directed mutagenesis
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Revision as of 23:53, 26 July 2007 by PhillipDodson (Talk | contribs)
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- Application: Single point mutation in plasmid exceeding 8Kbp
- Time to complete protocol from DNA template:3 days (including 2 overnight incubations)
Lab time: | waiting time | method |
---|---|---|
2hours (set up PCR) | 4 hours (PCR) | stratagene kit manual |
30min (add dnp) | 1hour(digest) | stratagene kit manual |
1hour (transformation) | 1hour(NZY media) | stratagene kit manual |
1hour (plate out) | 16hours (grow overnight) | |
1 hour (pick) | 16hours (culture overnight) | |
3 hours (miniprep) | Wizard kit manual | |
1 hour (set up Digest) | 2 hours | |
1/2 hour (Load Gell) | 2 hours | |
1/2 hour (Photo) |
- Approximate cost of materials for 10 mutations: AUD $2,000.00
Primary & secondary Reagents Required including controls
- Statagene quickchange XL Kit (#reactions+1 control) (kit 200522 (30 reactions))
- Promega wizard miniprep kit (#colonies picked per transformation(say 4) x # transformations)
- 100uL of 10mM IPTG (for blue/white control plate)
- 100uL of 2% XGAL in DMF (for blue/white control plate)
- Per transformation:
- 2 x Agar plates with Amp at 100ng/ml
- 125ng of each primer designed as per stratagene requirements
- 10ng of dsDNA template in less than 40uL
- 2 X 14mL BD falcon tubes #352059
- 0.5mL NZY broth
- 4 x ordinary falcon tubes for growing colonies
- for confirmation:
- Digestion enzymes and buffers
- 1% agarose gel,ethidium bromide,loading buffer, 1kb+ marker
Equipement Required
- PCR machine
- micro cebntrifuge for PCR tubes
- Ice
- waterbath at 42 deg C
- 37 deg C incubator with shaker
- gel manufacture and elecctrophoresis equipment for confirmation.
Method including controls
References
- PhillipDodson 03:48, 1 July 2007 (EDT):Secondary Reagent Template 1/July/2007