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 |
Latest revision as of 23:11, 28 February 2013
Protocol
Notes
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