15 replies to this topic

[novagen]

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

[gebirgsziege]

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.

[novagen]

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

[rye]

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

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

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

[perneseblue]

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

If I purify the PCR product and then give it for sequencing .is that ok.

Yup perfectly fine.

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

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??

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:

or is there any shortcut to be confident of thre sequence prior to cloning.

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.

[novagen]

Hi all,
thanx a lot for all the replyers

[tyrael]

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

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 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.

=)

[perneseblue]

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

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.

=)

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

[Biog]

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

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

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.

=)

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

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.

[perneseblue]

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.

[Biog]

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

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.

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

[tyrael]

orite. thanks for the replies. =)

[Trof]

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.

[HomeBrew]

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

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.

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?

[why]

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...

Edited by why, 22 February 2009 - 10:10 AM.