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
- Yeah, but small inserts also means enormous molar ratios even for small masses of DNA. Personally, if I'm working with something so small that I worry about purification, I'd just order the insert as complimentary oligos and avoid the PCR/purification process all together. -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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