A simple agarose gel electrophoresis and gel isolation procedure to remove oligos, template, and select only the PCR product of desired size is sufficient for most sequencing applications. If your end goal is cloning, then I would recommend cloning into a plasmid BEFORE sequencing since your PCR mixture can be heterogeneous. If PCR mutations are present, you may not detect them by sequencing depending on their frequency (instead you'll get the population average). Since a clonal derived plasmid will derive from a single PCR product, you can be sure the sequence you get back is what you'll actually recover (no guarantee that this occurs if you sequence prior to cloning).

Hi!

Once your PCR is completed with expected size band, you have to clean the excess of primer dimer with Exo-SAP before going to set your sequencing PCR.

Exo - SAP, Exonulease and Shrimp alkaline phosphotase.

You have to add both to the PCR product as per the manufacture instruction incubate them at 37C for 30 miniutes and denature at 80C for 15-20 minutes then go for sequencing PCR reaction.

go through the links you get details

http://www.iwai-chem.co.jp/products/usb/exosap-it.pdf

http://www.nucleics.com/DNA_sequencing_support/exonucleaseI-SAP-PCR-protocol.html

Good Luck!!

A simple agarose gel electrophoresis and gel isolation procedure to remove oligos, template, and select only the PCR product of desired size is sufficient for most sequencing applications. If your end goal is cloning, then I would recommend cloning into a plasmid BEFORE sequencing since your PCR mixture can be heterogeneous. If PCR mutations are present, you may not detect them by sequencing depending on their frequency (instead you'll get the population average). Since a clonal derived plasmid will derive from a single PCR product, you can be sure the sequence you get back is what you'll actually recover (no guarantee that this occurs if you sequence prior to cloning).

another possibilities is using paramagnetic beads to purify your PCR product and also select the size of band that you want.

this method is accurate and it can give you good product of DNA.

I think there are a lot of type of paramagnetic beads available in the market.

and when you just need simple methods, agarose gel electrophoresis works okay (at least for my sample)

hope this help ;)

Agree with Daniel. Running your PCR products on the gel and then removing your DNA from the gel is usually enough.

I think best way is to clone your gene in TA vector and then isolate plasmid and give for sequencing by M13 primer.

QIAquick PCR purification kit

A tip: send it to a commercial sequencing service.

This may be not the “correct” answer you expect, but is a tip.

I have used, as many others mention, “gel extraction” after electrophoresis with both, columns kits (for example from QIAGEN) and simple phenol/chloroform extraction. I have used extensively “direct sequencing” after a column purification (to remove buffer and primers/dimers).

But now I simple send the crude PCR (20 uL of my real-time-RT-SYBR-PCR-“melt curve” unopened reaction) to a commercial sequencing service (MWG). You may think it is expensive, but not, it is actually by far cheaper (by a factor of 2 or 3 approximately). I don’t need any “preparative” PCR (the original, “screening” PCR reaches). I don’t do any extensive manipulation, keeping my time for other thing, lowering my error and repeat rates. I don’t need any other (expensive) kit and disposables. And very important for me: we have much less contaminations. Also, the quality of the sequences are usually a lot superior.

Lawrence Mok was right

Ariel Vina-Rodriguez

I agree with you. I sent my sample (crude PCR) to commercial sequencing service but it don't sequence this, because PCR product was very bad and little. I want to purify product and re-amplify.

Hi!

If you send them for commercial sequencing service they do ask for Exo-Sap clean as per my experience (some Korean company) and they too check the product once you send. So repeat the PCR either with DNA if you have or from the purify product. Hope you get good results. Once you do everything perfectly certainly you get good results.

Good Luck!!

After confirmation of band of interest, I would cut out the band using a sterile scapel blade and purify it using PureLink Gel Extraction Kit (Invitrogen, CA, USA) according to the manufacturer's recommendations and finally the purified sample along with the associated primers can be sent to a commercial sequencing service eg Source Bioscience (http://www.sourcebioscience.com/) for sequencing according to their sample requirements. Best wishes.

With partial(e.a. 1/10) of your PCR product for quality checking on a Agarose gel at first. If it is perfect, meaning that is no smear and no unspecific band showing up, you can use your leftover (9/10) of your PCR product for purifying with a PCR cleaning kit from QIAGEN or whatsoever other companies. The resultant DNA can be sent out for sequencing. However, if the PCR product is not perfectly amplified, you may need run a gel and then cut out your expected band and do purification using a gel purification kit from QIAGEN.

I go with the answer of Gao on DNA sequencing of PCR product

Weimin Gao, I agree with you. But my PCR product is bad...

First, check the PCR product on the gel and then decide what method to use for sequencing.

I always run the PCR product on analytical gel first, then, if the product is a single product and the lane is clean, I purify the remainder of the reaction using the Zymo Research Clean and Concentrate kit- very fast, reliable, and compatible with any further procedure (you can use TE or water to elute). If the product is not clean and/or multiple bands are present, the best way to go is gel purification, as Daniel suggested. I also use a Zymo kit to elute the product from the gel slice- this one is ZymoClean. It works better for me than the analogous Qiagen kit for downstream applications. You can send this directly for sequencing, or use for cloning. As Daniel stated, if you are planning to clone, it would be best to clone first and send out several isolates for sequencing.

what you mean by your PCR product is bad? multiple bands? smears? faint band of your PCR amplicons? You might want to optimize your PCR reactions steps, if you planing to do cloning with it.

If the band is pure, you can directly purify with a PCR purification kit.

If not, run on a gel, cut the right band out, and purify with a gel extraction kit.

Any of the commercial kits work well (Qiagen, Zymo, ...)

I purified the PCR product by purification kit. But the result in nanodrp about 3,4 and 4.9 ng/ul? I do not know this is enough for sequence or not?

I think it is more than enough for sequencing as my memory goes. You can be in touch with any of the sequencing labs for confirming the same.

Ayat AL-Laaeiby, Which purification kit are you use?

If you have one band on gel, this concentration is enough.