I ran a PCR reaction and got my desired band at 800 bp with other non specific bands (reaction vol 25 ul) after optimization I got 800 bp band clear (reaction vol 25 ul). Then I went for bulk amount of PCR (reaction vol 100 ul) so that I can purify the band, but the band disappeared and after running PCR two to three times now I am frustrated as the sample was very small and could not be retrieved again. I have checked all steps instead of getting 800 bp I am getting 100 bp band. I have only 2-5 ul of sample left what should I do?
Hi Nadia,
This is a general stochiometric problem that appears often during direct up scaling of reaction volume. You have optimized reaction for 25ul vol. Try to upscale it for 50ul instead of 100 ul directly. Otherwise, you may opt four reactions of 25ul and pool the amplicon. All the best.
Hi Nadia,
This is a general stochiometric problem that appears often during direct up scaling of reaction volume. You have optimized reaction for 25ul vol. Try to upscale it for 50ul instead of 100 ul directly. Otherwise, you may opt four reactions of 25ul and pool the amplicon. All the best.
Firstly, dilute you sample. If the optimised PCR conditions are robust a 4 - 5 fold difference in the concentratin of your template DNA shouldn't make too much difference, just increase the number of cycles to maximise yield.
Be aware of the change in thermal inertia with increased sample volume, I know our BioRad PCR machine requires the sample volume and I presume compensates the times of the cycle steps accordingly.
I must admit I tend to run multiple 25 uL reactions if I need to bulk up for troublesome digests etc rather than increase the sample volume for this very reason.
completely agree with Deepak and Marcus.
One other possibility if you still have the small volume pcr that worked well dilute it 1:100 and run a pcr with 10-15 cycles as a reamplification but be careful not to run too many cycles as every 10 cycles will give you 1000 times more product
Besides the previous opinions ( I agree with all of them), please check how many freezing-thawing cycles has suffered your material in case you are using the same aliquotes (that is the same tubes of dNTP's, Taq, primers etc...
Hi Nadia:
I will suggest you to assess the quality of your DNA template . Specially if you eluted your DNA in water instead of TE. You can run 1ul of your template on 0.7-1% agarose gel. If the quality of your DNA is good(no smearing), then run multiple PCR reactions of 25 ul. you can amplify the PCR product in a second round of PCR using nested primers.
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