Dear all,
I am trying to amplify a approx. 1700 bp DNA sequence by PCR, that I have amplified before by ddPCR. I recovered it as described by BioRad with chloroform. Concentrations were 0,5-3 ng/µL initially and I saw clear bands at the desired range on the agarose gel. At first I tried to amplify 2 µL of this product using the same primers as used for ddPCR. It did not work. In a new attempt I purified the recovered ddPCR product with ethanol precepitation or a clean-up filtration kit because we suspected inhibitors to block the reaction. Concentrations after purification were very similar to those measured before the purification. Again, I only could detect primer dimers in the PCR.
Does anyone have experience with amplifying a recovered ddPCR product?
- Generally speaking you require about 1:100 of your recovered PCR product for secondary PCR (reamplification)
- However, I worry about the very low conc of your initial PCR product suggesting sub ambient purification
- In my experience, although dependent on sample type, good amplifications should result in much more product than your are obtaining
- Thus, I would first try and increase the efficiency and thus yield of your purified amplicon by altering your initial PCR conditions, e.g.
Thank you for your advise!
Which concentrations did you obtain by recovering the PCR product? Which supermix did you use for the ddPCR? I used 2x QX200 ddPCR EvaGreen Supermix. Did you use several replicates or just one reaction for recovery? Did you do a clean-up with a filtration tube after ddPCR recovery?
I already did 1, 3, 4 and 5. I will test using more of my initial PCR product.
- There are no hard and fast rules where these issues are concerned; like much in life science
- I would be guided therefore by (empirical) data
- Thus, use 1 1: 100 dilution (in duplicate) from your primary PCR reactions in your secondary PCR as described in my previous answer. No clean up
- If that does not work, I would column clean up and then take 1ul neat; 1ul 1:5 and 1ul 1:20 to put into your secondary PCR
- See which works best
- Badges
- Science method