Melbourne/Site directed mutagenesis
From 2007.igem.org
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| Line 16: | Line 16: | ||
| 1 hour (pick) ||16 hours (culture overnight) || | | 1 hour (pick) ||16 hours (culture overnight) || | ||
|- | |- | ||
| - | | 3 hours (miniprep) || || Wizard kit manual | + | | 3 hours (miniprep,and glycerol stock) || || Wizard kit manual |
|- | |- | ||
| 1 hour (set up Digest) || 2 hours || | | 1 hour (set up Digest) || 2 hours || | ||
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=====Primary & secondary Reagents Required including controls===== | =====Primary & secondary Reagents Required including controls===== | ||
*Statagene quickchange XL Kit (#reactions+1 control) (kit 200522 (30 reactions)) | *Statagene quickchange XL Kit (#reactions+1 control) (kit 200522 (30 reactions)) | ||
| - | * | + | |
| - | *100uL of 10mM IPTG (for blue/white control plate) | + | *For control plate: |
| - | *100uL of 2% XGAL in DMF (for blue/white 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: | *Per transformation: | ||
**2 x Agar plates with Amp at 100ng/ml | **2 x Agar plates with Amp at 100ng/ml | ||
| Line 40: | Line 42: | ||
**0.5mL NZY broth | **0.5mL NZY broth | ||
**4 x ordinary falcon tubes for growing colonies | **4 x ordinary falcon tubes for growing colonies | ||
| + | *for making glycerol stock | ||
| + | **600uL of 100%glycerol | ||
| + | **screw top cryotube | ||
| + | **Liquid nitrogen | ||
*for confirmation: | *for confirmation: | ||
| + | **Promega wizard miniprep kit (#colonies picked per transformation(say 4) x # transformations) | ||
**Digestion enzymes and buffers | **Digestion enzymes and buffers | ||
**1% agarose gel,ethidium bromide,loading buffer, 1kb+ marker | **1% agarose gel,ethidium bromide,loading buffer, 1kb+ marker | ||
Revision as of 00:02, 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 | |
| 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))
- 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)
- 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
