Smolke:Protocols/Insert prep: Difference between revisions
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==Protocol== | |||
*Prepare 5 x 100-ul PCR reactions for each insert | *Prepare 5 x 100-ul PCR reactions for each insert | ||
**400 ul water | **400 ul water | ||
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**250 ng plasmid | **250 ng plasmid | ||
**10 ul 1:10 Taq DNA polymerase | **10 ul 1:10 Taq DNA polymerase | ||
*After PCR, combine products from the five tubes, and run 5 ul on a gel to verify product length | *After PCR, combine products from the five tubes, and run 5 ul on a gel to verify product length | ||
*Purify and concentrate PCR products by phenol-chloroform extraction and ethanol precipitation | *Purify and concentrate PCR products by phenol-chloroform extraction and ethanol precipitation | ||
*Resuspend inserts to appropriate volume for digestion reaction | *Resuspend inserts to appropriate volume for digestion reaction | ||
==Notes== | |||
*Amplification rates: | |||
**Taq: 0.5-1 min/kb (YC). 1 min/kb (JKM) | |||
**Pfu: 2 min/kb (JKM) | |||
**PfuUltra: 1 min/kb (JKM) | |||
**PfuUltraII: 0.25 min/kb (JKM) | |||
**MutazymeII: 2 min/kb (JKM) | |||
*Size: I consider this ''massively'' oversized. In my experience, 50uL of PCR products (~3ug product) is more than sufficient for cloning. -Josh | |||
*Polymerase: I use Pfu (>500bp) or PfuUltraII (>~3kb) for any lengthy PCR that I intend to clone. -Josh | |||
*Magnesium: I have never felt a need to vary my magnesium concentration on a reaction-by-reaction basis. So I mix my Taq buffer using a standard amount of magnesium (2mM, personally), and save an extra step. The magnesium concentration given above (0.16mM) seems ''way'' too low. -Josh |
Revision as of 11:45, 15 October 2009
Protocol
Notes
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