How to Quantitate DNA

[premedstudent]

I ran a 25 micro liter PCR. I want to quantitate the DNA from my PCR. How do i do it? i know the basics but i dont know enough to be able to do it myself. I use gel electrophoresis and QiaQuick to purify the DNA from the gel after electrophoresis. Do i just add dye to the PCR reaction, and then add the entire PCR reaction to a well in the gel? how do i find out how much DNA i have in micro grams? thanks

[kiki06]

Hello there premedstudent,

I would use either a small amount of your pcr in a UV spectrophotometer to assay DNA using OD 260, or you could run a ladder like Lambda phage digest which has known amounts of loaded DNA.

The easiest and most quantitative (accurate) is the UV.

cheers

[premedstudent]

my lab does not use UV spec. so do i load the entire sample into the gel (gel electrophoresis)or only some? after that how do i know if a have enough? i need about 2 micro grams

[admin]

hello premed,
no I would load only some of it onto the gel. I would only use 1/5 of my pcr reaction or even less (i used about 5ul of a 50ul pcr reaction - even 25ul PCR rxns too).

Most people however UV spec before loading. If I didnt have access to one, just load 1/5 or more of your pcr reaction and you should see something if the PCR worked.
Buy the Lambda ladder digest which has DNA amounts to analyze.


However, make sure that you have enough extra pcr reaction for whatever you want to do later. I would optimize the pcr fully, then pcr a few tubes to use for gel and later preps.

That way you could use most of one tube to do a gel or two for publication purposes. Sometimes gels dont always turn out so pretty... (i usually store most of my leftovers in the -20 for this purpose)

cheers

[Priyanka]

I know its a liltle difficult for you to find a uv spec for quantification but that method would make your life really comfortable..(saving a lot of your sample and time)... otherwise ...Try loading 5 microliter of the reaction into the gel with lamda digest (like explained above by admin...) But if its a very important reaction PLZZZ do the at least one round of PCR of your PCR product before hand so that you have something to bank upon and this would also increase the amount of the elute as well...
All the Best...
Cheers