Smolke:Protocols/Insert prep: Difference between revisions

Revision as of 11:45, 15 October 2009

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Prepare 5 x 100-ul PCR reactions for each insert
- 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

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

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+{{Smolke_Top}}
+==Protocol==
 *Prepare 5 x 100-ul PCR reactions for each insert
 **400 ul water
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 **250 ng plasmid
 **10 ul 1:10 Taq DNA polymerase
-*Taq amplifies at 1000-2000 bp/min, so adjust extension time accordingly
 *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
+==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