Matt Gethers/CRI, Thailand/Labwork/PCRs/Screening for Presence and Directionality of HmgR Upstream Fragment in pUC18/calculations
Length of region including the upstream fragment of HmgR and the region between the PstI site and the M13 for primer binding site
Distance between BT1033 site and M13_rev site = 636 bp (see Vector NTI file).
636/1000 = .636 kb. 0.636kb*60 seconds/kb = 38.16 seconds. I've had a little trouble going with exactly the amount of extension time needed, so I'll add 10 seconds to the extension time (say 50 seconds).