For the removal of PCR inhibitors you can use DNA Clean-Up kit is very effective for the removal of PCR inhibitors that are commonly found in extracted DNA samples. For more details you can read the papers below;

Article A comparison of four methods for PCR inhibitor removal

Article PCR inhibitors – Occurrence, properties and removal

Article Efficient removal of PCR inhibitors using agarose-embedded D...

agree with @ Ibrahim Bala Salisu

regards

Dear Manan Mitra,

As suggested, it might be important to do an extra clean up step to get rid of any excess EDTA in your sample which might inhibit the PCR reaction.

Alternatively, you can look into adjusting the MgCl2 concentration in your PCR reaction (especially when working with blood samples containing EDTA).

Have you tried using Phusion blood direct PCR kit (http://www.thermofisher.com/order/catalog/product/F547S). The kit is said to work in the presence of different anticoagulants, with a polymerase that functions very well even in the presence of inhibitors found in blood.

I used the kit to work on mouse blood samples (collected in EDTA coated tubes). I did not perform a DNA extraction step, and used the blood directly in the reaction as per manufacturer's instructions. Though the kit is slightly pricey, it works very well, and saves cost and time of extracting DNA from your blood spots.

Good luck!

column clean ups or ethanol precipitation will help but first try just diluting the dna which will also dilute the inhibitors and sometimes less dna and less inhibitor means more pcr product. Add more MgCl2 as often inhibitors remove Mg so just try more Mg and double dilutions of the dna. If that fails then consider some of the KOD genetically modified polymerases that are active in dirty samples then clean up is not needed at all

Washing of DNA with ethanol may work, also use less template volume because more template may contain more inhibitors,

Hello Manan,

Your DNA quality seems fine and kits generally give pretty good quality products, however, your DNA quantity is very low.

I have a few questions;

What are your PCR reaction and cycling conditions? Can you show us a gel?

Have you successfully amplified PCR products from other samples using the same PCR protocol?

How do you quantify your DNA? A nanospec can give you the quantities you mentioned without any DNA in your sample.

Have you tried to amplify any housekeeping genes? If you can't get a housekeeping gene to amplify then it's likely you don't have DNA.

What are you eluting your DNA with? If you're using Elution Buffer (TE buffer) then large volumes can inhibit PCR itself, I've found. I think from your question you added 40 ng total DNA to your reaction, that would be 20uL of elution buffer (in the 2ng/uL sample), I think this is your issue as 40ng is plenty of DNA given your A260/280.

Try eluting your DNA with warmed dH2O or nuclease-free H2O (55-60^oC), add 10 - 200ng to your PCR reaction but standardise your DNA template quantity if you can. Also, by eluting DNA with H2O you can add much more DNA template with your low quantities.

Let us know how it goes!

Good luck,

Daniel

Dear Manan,like other researchers explained ,I think all you need first is to clean the extracted DNA through 70% cold ethanol,also check if your nanospec is well standardized, so we'll be sure the purity ratio is correct,then you can dilute your template DNA more during the Preparation of the PCR master mix.

Dear Mitra

you can use DNA Clean-Up kit which is very effective for the removal of PCR inhibitors that are commonly found in extracted DNA samples.Regard

In my experience, with those data you are providing, you have likely no product there. I presume that you measured conc. using NanoDrop or similar, and such low absorbance in combination with such high 260/280 suggest rather little DNA, if any, and absorbance is measured for RNA contamination.

Sure, try using clean-up kits or the cheaper EtOH washing, but you should first enhance the amount of input material and only after worry about having a pure sample.

Please let us know of further development.

Good luck.