Hello. I performed PCR (used the Vazyme Phanta Max Super-Fidelity DNA Polymerase kit), ran the product (50 uL) on 1% agarose gel. Sometimes the band is of a normal size sometimes its thinner, (I have no clue why and how that happens). I cut the band out carefully and purify it (Magen HiPurify kit). I add 15uL ddH2O and quantify it via nanodrop. I get "negative" quantifications, for instance: -4.78 ng or -8.54 ng. I have done this experiment so many times that at this point, I am at a loss on how to proceed next. Can anybody please help?
Hello,
It may be that the PCR conditions are not optimized, or bad quality of the template DNA. However, If low concentration is the problem, you can easily re-amplify your first PCR product by adding something around 1 uL of it to your PCR Master mix. Set up as many reactions as you need, then mix them and purify (or viceversa). That should solve the low concentration trouble. On the other hand, what are you using as blank? I've never used the purification kit you are using, but It could be that you are using an erroneus blanking solution. You should try using your elution buffer mixed with ddH2O in the same ratio that your purified product has. Good luck.
Adán Valenzuela Castillo I don't use the elution buffer to elute the DNA. I use ddH2O. So the blank i use is ddH2O. Thank you for the suggestion of re-amplifying my amplicon. I will give that a shot. Thank you.
Do you elute at room temp or at 37-50? I get higher yield when I use a little heat. Also, when I've gotten negative readings I always try reading it the next day and the majority of the time it rights itself and becomes positive (still a low value, but not negative!).
@catherine i elute at room temperature. I will try raising the temperature. Thank you for the suggestion
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue