I have amplified my samples using ITS1F and ITS4 primers. The gel has a faint band under the main band, the PCR settings are right as they work on other samples. The faint band is the same as the main band as I have sent them both for sequencing.
I did a PCR, then did a 1:10 dilution and did another PCR on the dilution using 1 ul of diluted product and 0.5 ul of diluted product in 50 ul reaction. My supervisor says I should mix both reactions together, then do something to purify the samples so I only get the main band to send off for sequencing. It doesn't involve a kit or running any more PCR, apparently it's really simple!
Does anyone have any suggestions?
Hi Caroline.
One thing you could try is: Run the gel and then physically excise the main band from the gel (e.g. using a blade) and extract the DNA from that slice of gel that you cut out. You can use a kit, such as the Qiagen QIAquick gel extraction kit to extract the DNA from the excised gel slice. You could then do another PCR run on the gel-extracted DNA band and sequence that. This should then ensure that only the main band gets sequenced (and nothing else). I have tried that a few times and it worked well for me. (Frankly, I don't remember whether I amplified the gel-extracted DNA or whether I sent it off to sequencing directly, but either way should be ok.)
I hope this helps.
Cheers, Oliver
It's NGS so I have to send off the pcr product, apparently the simple thing to do to purify it doesn't involve a kit or another pcr.
If your aim is just to get rid of primers and leftover dNTPs, then you can perform a PCR product clean-up reaction. For that you could use, for example, Exonuclease & Shrimp Alkaline Phosphatase (which can be purchased separately or as one set of enzymes, called 'ExoSA-IT). Alternatively, you could do a clean-up using Saphadex columns.
However, if more than one band appeared on your gel, and you want to ensure that only the "main band" gets sequenced (and not other faint bands), then I would suggest the method I described in my last comment. This would not require any ExoSAP-IT or Sephadex cleanup because the excised gel pieces should only contain the amplified DNA--the leftover primers would be separated on the gel.
For that method I don't think it matters whether it is NGS or Sanger or any other method of amplicon sequencing. If I understood you correctly, you want to sequence only the main band on your gel (i.e. the PCR product) and nothing else.
For the suggested method: When you cut out the band from the gel--you obtain the PCR product you want to target (plus a little bit of agarose gel)--I am assuming you run your PCR product on the gel and not genomic DNA). So once you excise the band from the gel, you can then purify the excised PCR product (band) from the gel using a kit such as QIAquick. You can subsequently do another round of PCR on the gel-excised purified PCR product, which should only amplify the region you want (i.e. the main band that you cut from your gel). That is because the primers have nothing else to anneal to (there should hardly be any genomic DNA in your gel), except the excised region that shows up as the main band on your gel.
As I mentioned, I have done that a few times, and it worked well for me. One more tip, in case you decide to try that method: Prepare a gel with low agarose concentration (e.g. 1%) because the agarose can interfere with the downstream process. Ideally, you want to use low-melting point agarose, but that is much more expensive than regular agarose, and gel purification with the suggested kit seems to work just fine with standard agarose as well.
Cheers, Oliver
I agree with Oliver. Cutting the gel with main band and doing the purfication with Qiagen QIAquick gel extraction kit worked for me too. But Oliver Manlik , do you have any experience about long length PCR products? I mean for those over 20kbp, I think Qiagen kit won't work.
Dear Sijia Xu,
no, I don't have any experience with such long amplicons.
I agree--I doubt that this method would work on really long PCR products.
Cheers, Oliver
We use Exo-SAP-IT to purify PCR products:
https://www.thermofisher.com/us/en/home/life-science/sequencing/sequencing-kits-reagents/exo-sap-it-express-pcr-cleanup-reagents.html?gclid=Cj0KCQiA-c_iBRChARIsAGCOpB36BeOq5rIn1mO7mc10ztgb8zRcRZpS3f43_qFoOCObIPzIk3nQk0kaAsxuEALw_wcB&s_kwcid=AL!3652!3!200508473977!p!!g!!exosap%20it&ef_id=Cj0KCQiA-c_iBRChARIsAGCOpB36BeOq5rIn1mO7mc10ztgb8zRcRZpS3f43_qFoOCObIPzIk3nQk0kaAsxuEALw_wcB:G:s&s_kwcid=AL!3652!3!200508473977!p!!g!!exosap%20it
very quick and easy.
You may try to purify PCR products first (instead of diluting them) and then run a gel on purified samples - the band should be better.
Hope that helps. Good luck!
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