Hello everyone. Recently I faced a situation, and would like to ask how to overcome it.
Vector : pENTR
Insert : 600 bp
I got many colonies and my colony PCR (with Gene-specific primers) came out positive. I sequenced all of them and failed.
I repeated the PCR with purified plasmid of those colonies individually with
1) Pair of gene-specific primers
2) Pair of vector-specific primers
I got the amplification with both the pairs. But unfortunately the sequencing failed again. I tried to send as PCR product as premix with the vector specific primer and yet again the sequencing got failed.
Please help me out with this since the KIT is very much costly I cannot afford to do so many reactions again and again.
can you attach a .abi sequencing file from 1 of the failed sequences please Anamika Singh .Sometime they give a clue to why the reaction failed. I assume that the pcr bands that you sent for sequencing were the right size. A picture of the checker gel for the pcr amplifications would also help
What company are you sending your samples too? Because everything else seems to be alright as you say.
Our of curiosity (because that was my one-time mistake). You did label the plasmid and the primers corrtly for them? Because I one time labeled them all as "PCR"-samples and not some as primers.
Dear Anamika
what do you mean for "sequence failed"?
Bad sequencing quality? or sequence different from expected?
One possibility that in my experience a possible problem that may happen in TOPO cloning is the INSERT insertion in reverse direction and you will not able to discriminate with PCR screeening using
1) Pair of gene-specific primers
2) Pair of vector-specific primers
You can easly check it by using 1 vector specific primer and 1 vector specifi primer or by looking carefully to the sequencing files.
However, I suggest to you to try PIPE cloning as alternative to TOpo cloning, is more cheap and more reilable, from my point of view.
You can find more information about it on my blog: ProteoCool ( http://proteocool.blogspot.com/ )
good luck
Manuele
Anamika Singh actually we can't rely on colony pcr for 600bp gene cloning due to its small length we may get non specific amplification, same happened with me for 508bp length gene , i got positive colony pcr with my gene specific primers but after double digestion i didn't found any fallout.
the best way to check wheather u have a positive clone or not is by double digestion and sequencing only...
Thanks
Thanks to you all for your valuable answers.
I would like to add one more thing that now I am getting the expected sequencing result in frame by sending the PCR product using gene specific primer.
But my concern is that why am I not getting the result using vector specific primer.
Is your no template pcr control negative with the gene specific primers ?
- Badges
- Science method