problem with cloning and sequencing. - (Oct/08/2007 )

Ihave recently embarked into the field of cloning. I have been trying to make a gene with FLAG tag at the amino terminus. I PCR (expand high fidelity PCR kit) amplified the ORF of my gene from the cDNA (purchased) and included hindIII (5 prime) and BamH1 (3 prime end) so that i could clone it into the vector. I ensured that the sequence of the ORF is 100% identical with the published sequence. I run the PCR product on a gel to make sure there is a good amount, purify, digest and gel purify both the PCR product and vector. The vector was also dephosphorylted prior to ligation. Used the pFLAG CMV2 vector. After ligation (at room temp for 30 minutes using the rapid DNA ligation kit from roche), I immediately transform 50-100 uL of competent DH5alpha cells with 2 ul of ligation reaction (1:2 and 2:2 work well for me). I do see decent number of colonies and check for the presence of insert by digesting with HindIII/BamHI. I send only those clones for sequencing which seem to have both my insert (1569 bases, runs slightly below 1.6kbp mark) and vector (4.7 kbp). I do see my FLAG tag, my RE and starting 20 bp's or so. Then I get sequence only from 220 bp till the end of my gene.
I have tried this many times. Even tried with different RE's. I am not sure how to proceed next or how to troubleshoot.

The first 200 bp's is extremeyl GC rich. I dont know if this is causing problems. I am going to sequence my PCR product just to make sure I am starting with the correct sequence even though, it seems to run around the expected region.

If someone has experienced this or can explain this, I would really appreciate it.

Thanks.

Priyas

.

Ihave recently embarked into the field of cloning. I have been trying to make a gene with FLAG tag at the amino terminus. I PCR (expand high fidelity PCR kit) amplified the ORF of my gene from the cDNA (purchased) and included hindIII (5 prime) and BamH1 (3 prime end) so that i could clone it into the vector. I ensured that the sequence of the ORF is 100% identical with the published sequence. I run the PCR product on a gel to make sure there is a good amount, purify, digest and gel purify both the PCR product and vector. The vector was also dephosphorylted prior to ligation. Used the pFLAG CMV2 vector. After ligation (at room temp for 30 minutes using the rapid DNA ligation kit from roche), I immediately transform 50-100 uL of competent DH5alpha cells with 2 ul of ligation reaction (1:2 and 2:2 work well for me). I do see decent number of colonies and check for the presence of insert by digesting with HindIII/BamHI. I send only those clones for sequencing which seem to have both my insert (1569 bases, runs slightly below 1.6kbp mark) and vector (4.7 kbp). I do see my FLAG tag, my RE and starting 20 bp's or so. Then I get sequence only from 220 bp till the end of my gene.
I have tried this many times. Even tried with different RE's. I am not sure how to proceed next or how to troubleshoot.

The first 200 bp's is extremeyl GC rich. I dont know if this is causing problems. I am going to sequence my PCR product just to make sure I am starting with the correct sequence even though, it seems to run around the expected region.

If someone has experienced this or can explain this, I would really appreciate it.

Thanks.

Priyas

.

Hi.

I think I was not clear about my problem in the message. When I sequence my clone (tried several clones), I get complete alignment till about first 20-40bp's and then from 220 bp's to the -end of my gene. But the middle 180 bps are missing. I just dont get them in the sequencing reaction. I have tried sequencing from the vector into the gene, from the middle to end of my gene, and also with reverse primers. But no use. Still that 180 bp's are missing.

I am trying restriction mapping now to make sure that part of the gene is there. It is not going very well too.

Thanks for you message.

if you have any additional inputs, please do share them.

Is the PCR product of the correct length? You could be correctly cloning and sequencing a product which was wrong. If it is wrong, then try adding 5% betaine to your PCR reaction to make high GC PCR work better.

If your PCR product is correct, then I'd think about recombination in your cloning strain. You might think about cloning into the SURE or STBL2 cloning strains, which are designed for stable cloning of troublesome inserts.

Thank you, Phage434.
I will try adding 5% betaine. Hopefully it helps.

I am guessing the PCR products validity based on its size in a 1% agarose gel. I am going to sequence my PCR product now just to be certain.

Thanks again.

it is not possible that you have a sequence read till intial 40 bp and then jump to 220bp of the cDNA in any circumstance.It could be possible the cDNA which you are clonig may itself be devoid of this 180bp.Why dont you confirm by digesting the cDNA with enzymes flanking this region such that you get a release which may confirm whether the cDNA you got is variant or not.

cloned, raises a point. So I am kind of confused. Do your sequence traces suddenly crash, so you have a blank space 200bp long. Or does the sequence trance looks normals, (if the sequencing primer starts from the front end of the gene)only the first 200bp is strangely missing.

So is the first 200bp present? And the sequencing won't read it. Or is the 200bp missing. And the sequence trances look normal

When I purchased the cDNA, I cloned the entire gene and I got complete ORF. Everything looked ok.
When I sequence my FLAG tagged ORF, i am missing 180 odd bases and the missing region shifts. Regions after 40bp is missing in one sequence while after 60 bp is missing in another. I usually get complete sequence from the 260th bp till the end of my gene.

Hence I am perplexed since it was there in my starting product. Digesting the cDNA with enzymes in this region is good suggestion.

Will try that. Meanwhile am waiting for ehst equence results of my PCR product.

I appreciate your suggestions.


Thanks.

This is a long shot, but just to make sure. Are you determining this missing region by actually comparing sequence, or by using Blast? If Blast, then you should be aware that the "Filter" feature of Blast will delete regions of your sequence prior to doing and reporting the match. Personally, I find this behavior completely obnoxious, but apparently others like it.