Smolke:Protocols/Insert prep

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(Protocol)
(Protocol)
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*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
 +
*After digestion, gel extract insert to remove the plasmid template
==Notes==
==Notes==

Revision as of 17:32, 15 October 2009

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Protocol

  • Prepare 5 x 100-ul PCR reactions for each insert (see notes below on reaction volume)
    • 400 ul water
    • 50 ul 10x Taq buffer
    • 16 ul 5 mM MgCl2
    • 10 ul dNTP
    • 10 ul Fwd primer
    • 10 ul Rev primer
    • 250 ng plasmid
    • 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
  • Purify and concentrate PCR products by phenol-chloroform extraction and ethanol precipitation
  • Resuspend inserts to appropriate volume for digestion reaction
  • After digestion, gel extract insert to remove the plasmid template

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
    • I think this depends on what you're cloning. My inserts tend to be <150 bp, which means getting 3 ug of product requires significantly larger PCR volumes. Smaller inserts are also more difficult to purify without loss downstream, hence the large volume that I generally use. --YC
  • 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
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