Hi,

Residual ethanol might inhibit enzymatic reactions.

Ramesh

I am not sure it was caused by ethanol residue remains in your amplicon stock cube. How much gDNA concentration did you yield after extraction? And how much working concentration did you employ in PCR master mix?

The smear you got might be the effect of hi gDNA concentration. If so, try to dilute your PCR product in ddH2O by ratio of 1:100 or less. Then perform nested-PCR with the diluted product as amplicon.

Best.

Hi Ramesh

As you know ethanol is one of the PCR inhibitors. But in this case I am not sure about that. The smear may because of hi genomic DNA in the PCR samples, AS Danny mentioned. Try it again with diluted DNA sample. I hope it goes well.

Good luck

This little bit of ethanol should not make a difference in your PCR and should not result in a total smear. Check the amount of DNA you put on your gel and how much DNA did you put into your PCR? I normally put 50 ng DNA into a PCR.

If you washed with 70% EtOH, keep in mind that most of the volume you saw which was left before adding the TE, was most likely water for the greatest part since the EtOH evaporates faster. Next time I suggest to be more patient to prevent these kind of suspicions though ;) I always dry my pellet in a heating block at 37 degrees C, it's very fast.

As for your question, I agree with the earlier suggestions to dilute your DNA.

Dilute it 100x, or even 1000x (or both). Extreme DNA concentrations, which could explain your smear, can also inhibit your PCR. Diluting will take care of that problem (as well as potential ethanol contamination).

Hi again,

I want to know what happened after the diluting part. If you try it, will be nice to share the result.

Best.

Thank you everyone for your valuable time.

As you guys suggested, I diluted my DNA in five different concentrations (as such, 1:5, 1:10, 1:50 and 1:100) but none of these amplified.  I pulled out few samples, treated with RNase A, precipitated with 95% ethanol, washed once with 70% ethanol and re-suspended in water. I could amplify only few of these!!

Still, I could notice that, some of the pellets are not dissolved yet.

I forget to mention in my previous post, 260/230 ratio was around 4 in my original DNA, I think it is too much. But after this treatment, it dropped to around 1.5.

Planning to repeat my extraction.

Thank you once again.

hi. at first you must get absorbance ratio260/230 that must be greater than 2. if it is lesser than 2 you must add ethanol 70% and centrifuge tube. then discard supernatant and inverse tube until remove alchole. but in most case you must repeated your procedure.

Hi , did you try purifying your Genomic DNA after isolation ,

good luck

PCR didn't work out probably because there is too much DNA in the sample and the smear is the DNA purified as you run a gel only with DNA. If the 260/230 ratio is 4, it's a high value, which means the ratio of pure DNA comparable with other compounds in the solution is high, probably due to high DNA concentration. The ratio of 2.0 - 2.2 indicates purity of DNA, and your value is 4.0, so there is a disproportion between your DNA and other components in the solution. This fact is in agreement with the smear result in electrophoresis (what you saw was DNA and no PCR product). Ethanol is an inhibitor of PCR, so its presence may also have been the cause of your PCR failure, in addition to the high DNA concentration.

Complementing, the presence of ethanol does not alter 260/280 and 260/230 ratios values.

Hi there,

I read this question and the answers today and though this question was asked 3 years ago, I nevertheless want to add in answer:

I recommend to use ddH2O for dissolving your DNA pellet if you want to do PCR later on because EDTA (TE buffer = Tris and EDTA) is a strong PCR inhibitor. EDTA complexes divalent cations such as Mg2+ but Mg2+ is an important cofactor for polymerases.