Melbourne/Site directed mutagenesis
From 2007.igem.org
(Difference between revisions)
Line 16: | Line 16: | ||
| 1 hour (pick) ||16 hours (culture overnight) || | | 1 hour (pick) ||16 hours (culture overnight) || | ||
|- | |- | ||
- | | 3 hours (miniprep,and glycerol stock) || || Wizard kit manual | + | | 3 hours (miniprep,and glycerol stock) || || Wizard kit manual -->next mutation cycle--> |
|- | |- | ||
- | | 1 hour (set up Digest) || 2 hours || | + | | 1 hour (set up Digest, make gell) || 2 hours || |
|- | |- | ||
|1/2 hour (Load Gell) || 2 hours || | |1/2 hour (Load Gell) || 2 hours || | ||
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*for confirmation: | *for confirmation: | ||
**Promega wizard miniprep kit (#colonies picked per transformation(say 4) x # transformations) | **Promega wizard miniprep kit (#colonies picked per transformation(say 4) x # transformations) | ||
- | **Digestion enzymes and buffers | + | **Eppendorf, Digestion enzymes and buffers. |
- | **1% agarose gel,ethidium bromide,loading buffer, 1kb+ marker | + | **1% agarose gel,ethidium bromide,loading buffer, 1kb+ marker. |
Line 60: | Line 60: | ||
*37 deg C incubator with shaker | *37 deg C incubator with shaker | ||
*gel manufacture and elecctrophoresis equipment for confirmation. | *gel manufacture and elecctrophoresis equipment for confirmation. | ||
+ | *Camera | ||
=====Method including controls===== | =====Method including controls===== | ||
# | # |
Revision as of 00:08, 27 July 2007
<Return to list of protocols> <Team home page>
- 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 |
---|---|---|
2 hours (set up PCR) | 4 hours (PCR) | stratagene kit manual |
30min (add dnp) | 1 hour(digest) | stratagene kit manual |
1 hour (transformation) | 1 hour(NZY media) | stratagene kit manual |
1 hour (plate out) | 16 hours (grow overnight) | |
1 hour (pick) | 16 hours (culture overnight) | |
3 hours (miniprep,and glycerol stock) | Wizard kit manual -->next mutation cycle--> | |
1 hour (set up Digest, make gell) | 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))
- For control plate:
- 100uL of 10mM IPTG (for blue/white control plate)
- 100uL of 2% XGAL in DMF (for blue/white control plate)
- 1 x Agar plates with Amp at 100ng/ml
- 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 making glycerol stock
- 600uL of 100%glycerol
- screw top cryotube
- Liquid nitrogen
- for confirmation:
- Promega wizard miniprep kit (#colonies picked per transformation(say 4) x # transformations)
- Eppendorf, 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.
- Camera
Method including controls
References
- PhillipDodson 03:48, 1 July 2007 (EDT):Secondary Reagent Template 1/July/2007