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Revision as of 23:53, 26 July 2007 (view source) PhillipDodson (Talk | contribs) ← Older edit		Revision as of 23:56, 26 July 2007 (view source) PhillipDodson (Talk | contribs) m Newer edit →
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	!Lab time:    !!waiting time !! method		!Lab time:    !!waiting time !! method
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-	|2hours (set up PCR)  ||4 hours (PCR) || stratagene kit manual	+	|2 hours (set up PCR)  ||4 hours (PCR) || stratagene kit manual
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-	|30min (add dnp)  ||1hour(digest) || stratagene kit manual	+	|30min (add dnp)  ||1 hour(digest) || stratagene kit manual
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-	| 1hour (transformation)  ||  1hour(NZY media) || stratagene kit manual	+	| 1 hour (transformation)  ||  1 hour(NZY media) || stratagene kit manual
	|-		|-
-	| 1hour (plate out) || 16hours (grow overnight) ||	+	| 1 hour (plate out) || 16 hours (grow overnight) ||
	|-		|-
-	| 1 hour (pick)  ||16hours (culture overnight) ||	+	| 1 hour (pick)  ||16 hours (culture overnight) ||
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	| 3 hours (miniprep)  ||      || Wizard kit manual		| 3 hours (miniprep)  ||      || Wizard kit manual

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Revision as of 23:56, 26 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)		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