Hello all,
Previously after completing my PCRs, their electrophoresis gels indicated all PCRs had primer dimers.
I am having difficulty ridding my PCRs of dimers as well as anything 100 picomoles/ul. Because I will be sequencing with an Ion Torrent, I need dimer concentrations to be less than 100 picomoles/ul.
However, some PCRs that were purified twice showed no dimers on the bioanalyzer with decent concentrations of the targeted DNA (i.e., 200bp-400bp). I am wondering if my PCRs initially have so much dimers that it overwhelms the Ampure beads during the first purifications? And if it is reasonable to complete multiple Ampure purifications to remove remaining dimers?
What are your thoughts and recommendations?
Thank you,
Gary
try using less primer and a hot start enzyme to minimise the p-d. This may work if the pd is being produced fresh in each reaction. Not so effective if you have pd contamination. Gel separation then a single gel purification may be an option and will remove pd very effectively. If you are running large numbers of samples consider designing a better primer pair
consider also touchdown pcr
I recommed to use less primers, up to 0.3 uM. A good opcions before sequencing is purify the PCR product with ExoSAP-IT PCR Product Cleanup Reagent, it works very well.
Thank you all for your answers.
Carlos, I've previously considered using ExoSap. Yet, please correct me if I'm mistaken, but isn't ExoSap only effective at removing ssDNA, and not dsDNA like dimers?
Using Thermofisher's "Multiple Primer Analyzer" tool, to my chagrin I recently found that some of my primers are likely forming self-dimers and cross-dimers. Unless I redesign my primers, I will likely continue to have dimers regardless of volume.
On the other hand, two of my PCRs (out of six) that underwent two Ampure bead cleanups at 1.0x concentration had no dimers. This includes one PCR product that had dimers after one Ampure cleanup.
Instead of 1.0x, I'm also testing use of 0.7x beads because my PCRs contain BSA which I've read may interfere with DNA binding to the beads. Beckman-Coulter tech support informed me that BSA concentrations up to 100 ng/ml shouldn't cause interference. However, my PCRs contain BSA of 500 ng/ml. Additionally, another PCR reagent I used is the detergent Tween-20.
Has anyone encountered interference of DNA-binding to Ampure XP beads from BSA and/or Tween-20 (or other detergents)?
Best regards,
Gary
Another possibility is to do a size selection step, like a Blue Pippen selection, after you are done with PCR. You could just have the sequencing company do this to your library before they sequence it. A Blue Pippen run normally costs just a few bucks (I think I paid 20 or 30 US last time).
You would just select for your expected amplicon size range and get rid of the very short primer dimers and extra adaptor sequences. Cheers
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