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

Hi all,
As Iam unaware about the sequencing process. I would like to clarify a simple doubt.
Can the PCR product be sequenced before cloning it into the vector using the gene specific primers???

Thanx a lot

If you remove excess primers, enzyme, dNTPs prior sequencing AND if you have a good PCR product (app. 10ng DNA / 100 bp seq) form ONE sequence (no contaminatin seq) you do not need to clone it.

But you should think that the first 50bp or so will not be sequenced. So if you need you sequence full length you should do it in both directions.

hi gebirz

If I purify the PCR product and then give it for sequencing .is that ok.
becoz I feel it would be better to sequence the pcr product and then clone it rather than troubling so much for cloning and getting the non specific sequence. what do you say?? or is there any shortcut to be confident of thre sequence prior to cloning.

Thanx a million times

novagen on Jan 26 2009, 09:21 AM said:

You might also try the RE digestion and check the digested bands

novagen on Jan 26 2009, 08:21 AM said:

Yup perfectly fine.

novagen on Jan 26 2009, 08:21 AM said:

If you are using proof reading DNA polymerases, designed your primers well (avoiding multiple binding of the primer to the template DNA), obtain a PCR products of the right size, this isn't a problem.

It should also be noted that sequencing only gives the average sequence of the PCR product. Not the sequence of any one PCR product. So you will have to sequence the PCR product a second time, once you have cloned it to make sure this one molecule that you have cloned is correct.

novagen on Jan 26 2009, 08:21 AM said:

As mentioned there is restriction digest. There is also southern blot, where your probe your large PCR product with DNA probes which can be subsection of your larger product, or DNA from another species which is similar. There is PCR analysis, where you use primers to PCR amplify subsections of your larger PCR product to show that all those subsections are present.

But again note, these method only look at the average PCR product, not a particular molecule. So you must recheck the PCR product once it is cloned into a vector.

Hi all,
thanx a lot for all the replyers

gebirgsziege on Jan 26 2009, 06:55 AM said:

hi all. i assume that is so called "direct sequencing" , by which we just gel purified the PCR product and then send it for sequencing ?

however, regarding the removal of excess primes, enznyme, dNTPs, if u could come out with more input to share with ? i would like to know how to remove those. as far as i know, direct sequencing sometimes we get a noisy background in the result.. of course, the bestway is still to clone it into known vector but it took time. so, if you;re pleased to share, then thank you very much in advance.

=)

tyrael on Feb 21 2009, 07:22 AM said:

Gel purification or PCR clean up columns will do the job.

perneseblue on Feb 21 2009, 09:14 AM said:

Yet, I'm a little bit sceptic about the real necessity to purify PCR product prior to sequencing it, as sequencing reaction, similarly to a classic PCR, contains too a PCR mix (primer, dye, enzyme...) but we sequence with ! So, in my mind, purifying PCR product would not be necessary if the PCR product seems to be at the right size. But as mentioned above, sequencing after cloning is mandatory, depending on the subsequent aim, to check the clone integrity and the absence of mutation.

my lab initially didn't clean up the PCR products prior to the sequencing reaction. However we found that if we did (clean up), we got a much better success rate of obtained a sequence read.