From 2007.igem.org

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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
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