sequencing the pcr product - (Jan/26/2009 )

perneseblue on Feb 21 2009, 03:05 PM said:

Yes, it is always much better to use a cleaned product than a "mixed" one.

orite. thanks for the replies. =)

We use Qiagen kits for Gel Extraction (if we cut the desired band out of gel) or PCR purification (if there is a clear single product) and then sequence it, this is a standard procedure here, we search for mutations and we don't clone the product, just sequence it. Both protocols differ only in the first step, use the same columns (MinElute kits). And at the end we get about 15 ul of 10-50 ng/ul purified product ready for sequencing.

perneseblue on Jan 26 2009, 03:17 PM said:

This is a very important point; make sure you take note of it. Since you're cloning the product, there's not much value in sequencing the product before cloning it, because the sequence you get from the PCR reaction does not necessarily tell you the sequence of your ultimate clone, which I presume is more important to you.

Since you'll need to sequence your insert after cloning it anyway, is it necessary to also sequence before cloning it?

Can we get clean sequencing results without purifying it? I thought the excess primers in the PCR products would also "participate" in the sequencing reaction and synthesizing sequence, for example, from the other end, and thus causing the background noise...

why on Feb 22 2009, 07:09 PM said:

Many things can cause background noise, not HPLC-cleaned primers, unpurified cycle sequencing,...sometimes you can't even figure why, so I wouldn't try to put unpurified product to the reaction and expect no background. It's too expensive to let it fail on that.