We tried to specifically remove template DNA after PCR reactions. Because of unique application, combination of Dam methyltransferase together with DpnI did not work well. Combination of T4 PNK and Lambda exonuclease worked but it still digested 40-50% of the PCR product (non-phosphorylation products).
Do you have any other method to specifically remove template DNA after PCR?
Thanks,
Zhiwei
you didn't mention the sample or other details.. Please go through these link. This might help you.
https://www.researchgate.net/post/How_do_I_remove_eukaryotic_genomic_DNA_from_PCR_fragments
https://www.researchgate.net/post/How_can_we_use_PCR_product_as_the_template_for_doing_another_PCR_to_increase_the_target_one
Article An efficient method for purification of PCR products for sequencing
Hello
I am not sure due to lack of details, but this might be the answer: You may purify your specific PCR product from the rest of reaction components by agarose gel extraction.
Dear Zhiwei Chen
Before doing PCR, use the terminal transferase enzyme to put some A nucleotides at the end of your template, after PCR reaction, use of PA exonuclease is helpful for removing your template. If your template and product length are the same.
Mohammad Hashemabadi : It seems like an interesting idea. What is PA exonuclease? I tried searching but can't find it.
Thanks,
Kevin
You can use the size selection properties of AMPure XP beads. Depending on size of PCR product either retain the supernatant then purify by precipitation or a column like Zymo or Qiagen's PCR cleanup. The reverse size selection will ensure the large DNA products are bound by the beads and the small product much in excess will be in the supernatant. Another approach would be a double bead cleanup which is actually used in your companies Ampliseq products. You can find all sorts of info about size selection and Ampure beads. Below are just a few links that might be helpful.
http://core-genomics.blogspot.com/2012/04/how-do-spri-beads-work.html
https://www.neb.com/protocols/0001/01/01/size-selection-e6270
https://www.takarabio.com/learning-centers/nucleic-acid-purification/dna/rna-cleanup-and-extraction/nucleomag-ngs-clean-up-and-size-select?gclid=EAIaIQobChMIqubntt306AIVg5-fCh16Uw2fEAAYAiAAEgKE6_D_BwE
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