how to check primers - (Aug/25/2006 )
I have been trying to clone many genes at the same time, so I decided to go for blunt end cloning, as I had many problems with the Gateway-Invitrogen people and their products.
So, finally I got some positives, and when sequencing I see that the first codon (the ATG) is missing....and the final base from the last codon at the 3' end, is missing as well. The rest is as it should be.
I checked again the list from when I ordered the primers, and I didn't do any mistake. Several colonies I sequenced from the same clone, showed the same.
Do you think it is possible the company made a mistake when producing the primer or are you familiar with this artifact if it is so?
Now I will order new primers to another company, but I wonder what is this. Any light into this subject it is very much appreciated.
This has happened to me too. I had ordered oligos from MWG and had problems with getting positives. As later confirmed by the company (upon complaint), the sequence was wrong. According to the company's advice I ordered them "MWG-HYPUR" gel purified. And this time it worked. But this also depends on how long your oligos are...
What could also be, is that the sequence of your primers is ok (but still, let someone else check them!), but you haven't checked enough clones... sometimes it takes a little longer.
Sometimes companies tell u that errors could occur if primers r too long. I never understood y.
Thanks for your reply Jou. I was suspecting that, production problem. I had tried over 100 clones, and I got 10 positives, all with the same sequence. The rest, was clear the insert was not there, that's why I suspected the primers.
Pity, pity....so much work. Now I will try with new primers from another company. Mine was INVIT......never again.
Yours definitely sounds like a bad primer synthesis especially considering your insert ratio.
When sequencing you can sometimes find the same artifact.
If sequencing near the ends of your target fragment you'll get bad sequence traces at or near your sequencing primer. When sequencing, if possible, use a primer at least 50bp upstream of your target.
just an aside...