Smolke:Protocols/Insert prep

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 50 mM MgCl2
 * 10 ul 10 mM dNTP
 * 10 ul 10 uM Fwd primer
 * 10 ul 10 uM 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
 * 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
 * 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