problems with low concentration DNA templates for PCR - (May/29/2008 )
Dear all
i face a problem with the conc of DNA in my samples
that i try to measure the OD of my sample at 260 i found it was 0.003 so concentration of my DNA is 0.003 X 50X 600/5 (dil. factor) = 18 ng/ul
in order to do PCR i need template DNA at concentration of 50 ng/ul
how can i solve it??????
PLZ HELPPPPPPPPP
you could re-precipitate it and resuspend in a lower volume.
.003 is a very low, unreliable reading. don't expect to get a stoichiometric recovery.
i face a problem with the conc of DNA in my samples
that i try to measure the OD of my sample at 260 i found it was 0.003 so concentration of my DNA is 0.003 X 50X 600/5 (dil. factor) = 18 ng/ul
in order to do PCR i need template DNA at concentration of 50 ng/ul
how can i solve it??????
PLZ HELPPPPPPPPP
1. It also depends upon what DNA you have, if you have genomic DNA, PCR is less likely to work, if it is plasmid or PCR DNA, it may sail, if it is cDNA and your gene is in great abundance it will work.
2. You probably don't have to have 50ng/ul conc as a strict rule, in that case, you can use sample volume of upto 5% of PCR reaction (Edit: and sometimes upto 20%, if dissolved in ddH2O) without worrying about how much DNA template there is.
3. At your OD, it is possible that you have NO DNA at all or unreliable amount of DNA. You can run a few ul on mini-gel to see whether you have some DNA.
4. You can precipitate with glycogen, use the entire pellet for PCR if you can sacrifice all DNA.
5. Get / make other samples.
OD260 of DNA is unreliable and generally inaccurate at those concentrations. The most accurate OD260 is around 0.5. You don't need 50 ng to do a PCR, you can have much less than that. If you're worried about the concentration of your template, perform a few different PCRs with 1, 2, 4 uL of template, etc. Be aware though that if the PCR does fail, it may not be because of a lack of template. That can be a cause but there are many others too.
Thanks all for reply and help
At first this is genomic DNA extracted from around 1 ml of rat blood. i face many problem in extraction of DNA from this small volume of blood trying many methods but all that was waste of time so i decide to run agarose gel to see if there DNA in my samples or not ....... i saw that all my samples have DNA with a very obvious band even i take only 3 or 4 ul from each sample. so my supervisor when see it told me that those bands are very good for PCR. but i cann't know why do they give me those low concentrations at OD/260. i like to discuss my research problems here as i can think with you all............PLZ tell me what i can do in the PCR procedure before asking my supervisor???
Best regards.
The best thing for a PCR, especially when unsure but desperate for results, is to set up multiple conditions. I have a panel "PCR optimization buffers" with varying amounts of MgCl2, KCl and different pH of Tris. I set up the pcr with the same amount of template, primers, dNTPs, enzyme but with the different buffers. So far I've always been able to get a product from at least one condition. I would gladly send you the recipes for each buffer if you want. Otherwise, just give the PCR a chance with the typical reaction setup and see what you get. From that you can optimize if need be.