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how much template do i need for pcr?


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5 replies to this topic

#1 bubbletea27

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Posted 25 April 2013 - 02:06 AM

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.

#2 GNANA

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Posted 25 April 2013 - 03:08 AM

you can use around 20-40ng plasmid for PCR.
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#3 bubbletea27

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Posted 25 April 2013 - 03:22 AM

Yeah thing is i dont have a machine to quantitate the concentration. How much dp you usually use?

#4 GNANA

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Posted 25 April 2013 - 03:37 AM

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.
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#5 bubbletea27

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Posted 25 April 2013 - 03:47 AM

Think ill try that. Thanks~!

#6 mdfenko

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Posted 25 April 2013 - 04:45 AM

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).
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