Smolke:Protocols/Insert prep

Home        Contact        Internal        Protocols        Lab Members        Publications        Research       

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 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