Smolke:Protocols/Insert prep: Difference between revisions
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**50 ul 10x Taq buffer | **50 ul 10x Taq buffer | ||
**16 ul 50 mM MgCl2 | **16 ul 50 mM MgCl2 | ||
**10 ul dNTP | **10 ul 10 mM dNTP | ||
**10 ul Fwd primer | **10 ul 10 uM Fwd primer | ||
**10 ul Rev primer | **10 ul 10 uM Rev primer | ||
**250 ng plasmid | **250 ng plasmid | ||
**10 ul 1:10 Taq DNA polymerase | **10 ul 1:10 Taq DNA polymerase (Starting Mar 2013, we will use 5 ul of 1:20 Taq DNA polymerase)- YHW | ||
*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 | ||
*DpnI treat the PCR products to remove the template - add DpnI directly to the PCR reaction (0.5uL in a 50uL reaction is plenty). Incubate 1hr at 37C. -Josh | *DpnI treat the PCR products to remove the template - add DpnI directly to the PCR reaction (0.5uL in a 50uL reaction is plenty). Incubate 1hr at 37C. -Josh | ||
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****Ordering complimentary oligos would certainly be the least labor intensive. It becomes tricky when the insert is too long for accurate synthesis, which is usually the case with the miRNA and ribozyme switch sequences we typically use. --YC | ****Ordering complimentary oligos would certainly be the least labor intensive. It becomes tricky when the insert is too long for accurate synthesis, which is usually the case with the miRNA and ribozyme switch sequences we typically use. --YC | ||
*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 | *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. -Josh |
Latest revision as of 23:11, 28 February 2013
Protocol
Notes
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