Smolke:Protocols/Insert prep: Difference between revisions
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*Size: I consider this ''massively'' oversized. In my experience, 50uL of PCR products (~3ug product) is more than sufficient for cloning. -Josh | *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 | **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 | *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 | *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 14:33, 15 October 2009
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