how much template do i need for pcr? - (Apr/25/2013 )
Hi this question is probably really basic but i am new at lab and would be nice if someone can answer my question.
After doing miniprep, if i wanna do pcr how much volume do people usually take? The yield seemed good on the gel. I eluted with 40. The size of the plasmid is about 6k.
you can use around 20-40ng plasmid for PCR.
Yeah thing is i dont have a machine to quantitate the concentration. How much dp you usually use?
its hard to say, atleast one shd have an idea how much the expected conc. of your miniprep, so accordingly you can dilute.
if you think you hav a good conc from miniprep, i would say do a 1:10 dilution and use 1-1.5 microlitre for a PCR...assuming your miniprep yielded you around 250ng/microlitre.
Think ill try that. Thanks~!
you can run an aliquot on agarose with quantified size standards to get a rough idea of the concentration (and you can recover the plasmid loaded onto the gel).