Melbourne/Site directed mutagenesis
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**100uL of 10mM [[Melbourne/primary IPTG|IPTG]] (for blue/white control plate) | **100uL of 10mM [[Melbourne/primary IPTG|IPTG]] (for blue/white control plate) | ||
**100uL of 2% [[Melbourne/primary Xgal|Xgal]] in DMF (for blue/white control plate) | **100uL of 2% [[Melbourne/primary Xgal|Xgal]] in DMF (for blue/white control plate) | ||
| - | **1 x [[Melbourne/Secondary Reagent Agar Plates|Agar Plates]] with [[Melbourne/primary AMP|Ampicilin]] | + | **1 x [[Melbourne/Secondary Reagent Agar Plates|Agar Plates]] with [[Melbourne/primary AMP|Ampicilin]] at 100ng/ml |
| - | at 100ng/ml | + | |
*Per transformation: | *Per transformation: | ||
| - | **2 x [[Melbourne/Secondary Reagent Agar Plates|Agar Plates]] with [[Melbourne/primary AMP|Ampicilin]]at 100ng/ml | + | **2 x [[Melbourne/Secondary Reagent Agar Plates|Agar Plates]] with [[Melbourne/primary AMP|Ampicilin]] at 100ng/ml |
**125ng of each primer designed as per stratagene requirements | **125ng of each primer designed as per stratagene requirements | ||
**10ng of dsDNA template in less than 40uL | **10ng of dsDNA template in less than 40uL | ||
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**600uL of 100% [[Melbourne/primary glycerol|Glycerol]] | **600uL of 100% [[Melbourne/primary glycerol|Glycerol]] | ||
**[[Melbourne/Screw Top tubes|Screw Top cryotube]] | **[[Melbourne/Screw Top tubes|Screw Top cryotube]] | ||
| - | **Liquid | + | **[[Melbourne/primary Liquid N2|Liquid Nitrogen]] |
*for confirmation: | *for confirmation: | ||
**Promega wizard [[Melbourne/Miniprep kit|Miniprep kit]] (#colonies picked per transformation(say 4) x # transformations) | **Promega wizard [[Melbourne/Miniprep kit|Miniprep kit]] (#colonies picked per transformation(say 4) x # transformations) | ||
**[[Melbourne/1.7ml microcentrifuge|eppendorf]], [[Melbourne/primary Restriction enzymes|Restriction enzymes and buffers]] | **[[Melbourne/1.7ml microcentrifuge|eppendorf]], [[Melbourne/primary Restriction enzymes|Restriction enzymes and buffers]] | ||
**1% [[Melbourne/Preparing an agarose gel|agarose gel]],[[Melbourne/primary EB|Ethidium Bromide]],[[Melbourne/Secondary Reagent TAE|Loading Buffer]], [[Melbourne/primary DNA marker|1kb+ marker]]. | **1% [[Melbourne/Preparing an agarose gel|agarose gel]],[[Melbourne/primary EB|Ethidium Bromide]],[[Melbourne/Secondary Reagent TAE|Loading Buffer]], [[Melbourne/primary DNA marker|1kb+ marker]]. | ||
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=====Equipement Required===== | =====Equipement Required===== | ||
Latest revision as of 11:34, 29 September 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 Ampicilin at 100ng/ml
- Per transformation:
- 2 x Agar Plates with Ampicilin 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 NYZ 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, Restriction enzymes and buffers
- 1% agarose gel,Ethidium Bromide,Loading Buffer, 1kb+ marker.
Equipement Required
- PCR machine
- microcentrifuge: 0.6ml
- 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
