My template DNA has high quantity of protein impurities I did PCR and get a good band after gel electrophoresis but I saw a smear in the gel
I have checked the DNA purity with nanodrop from the data I find that my template has high protein impurities the smear in gel electrophoresis support this theory
Now I want to send my products for sequencing but I want to know “what is the best purification technique for this kind of samples”
Hi Mushtaq, is there a proteinase step in your dna extraction procedure? Perhaps use a procedure/kit that includes this to help remove contaminating protein from your final extraction. Also, are you using column based purification? If the problem still persists you could always cut the good band from your gel, purify it and then measure before sending for sequencing.
Use gel purification to isolate your DNA product.
(a) Cut out the correct PCR product band from the agarose gel and extract the DNA from it using a product such as QIAquick Gel Extraction Kit
(b) PCR it up again. Check for impurities. Repeat gel purification technique until the band is clear on the gel. Then make one final gel purification of that band.
(c) Speak to the Sequencing service - ask them how they want the DNA prepared. If this means using a different clean-up kit, buy the one they recommend and use it.
Hi. If your DNA is large enough, yo can run it through centricon devices with the appropriate cut-off after treating your sample with proteases: the DNA should be retained and the protein fragments pulled down.
Also think about precipitate the proteins impurities through heat-shock (es: 80-90 °C), pull down them by centrifugation and finally recover your DNA-containing supernatant.
Two options you can try:
1. First is to treat your samples with proteinase K (Qiagen or Sigma) as instructed and then do DNA purification using conventional techniques such as phenol-cloroform method or by column.
2. Second by using DNA purification kit (for example Norgen or Zymoresearch) and be sure to wash well.
Either way run your purified DNA on the gel and check if it is okay.
Good luck!
I think Philip, Annie, and Zakaria gave the best answers
good luck
https://www.researchgate.net/post/Which_type_of_PCR_techniques_is_the_best_in_your_opinion?isAnswerFieldFocused=true
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