The weirdest primer ever - (Jul/20/2006 )
Hi. I tell you the story of this very weird primer which I order from a worldwide recognised company.
Sometimes I check my primers to see if the rehydratation process went well. I usually do this running a good amount in a gel, like this one:
Which is a serie of different primers 20ul of concentration 5uM. But recently because of some weird results in my PCRs, I decided to use a biophotometer to measure the stock concentration and what my surprise is that the third from the right doesn't show any absorbance at 260 whereas the fourth (and others) coincide quite accurately with the 100uM expected. Band size of the third one is between 50 to 100 bp.
I know that spectrophotometer measurements are not very realible for minimal amounts but I've used the same biophotometer for 6 different primers in one go and results are similar to the gel and the expected concentration (one differs from the 100uM expected but the band in the gel is also more faint). I've repeated three times getting the same result.
I rehydrate this one with TE together with many of other primers in one go and the rest of them worked really well. This primer is 33 bp because it has a tag.
Could anyone give me any idea about how this happens with the third primer from the right? How do I get so much light when the biophotometer indicates that there is nothing (I run a gel after the measurement getting identical result to the one above). Dimerisation? But I should get absorbance.
Please let me know what was wrong because this tiny mistake has made me lost about 1-2 months of work. Grrrrr.
Now I can see it.
If you say that the band is double the size, I would say that this is dimerization. But you should have absorbance, you're right! Maybe you should change the primers?
Yes, I'm gonna order them again and this time I will do the double check.
However what I want is to be able to explain to the rest of people (for example my supervisor)why I got that weird band. Besides, I got results during this month of PCRs with this primer. It gave some results quite close to those I expected (sometimes 100bp higher others below, depending on the conditions) and also hard to reproduce in the same conditions.
Does this give you a hint?
To be honest, I don't have a clue. ????
HI,
I think that if you suspect your primers, its best that you talk to the company that made them and ask them what wrong. And then, ofcourse, post your anwser here...
Gi
your primer might be degraded. how old is it? how do you store it?
did you use the primer stock or primer dilution you prepared earlier when loading your gel?
did you use the primer stock or primer dilution you prepared earlier when loading your gel?
It is only 2 months old and I stored at -20C. I used primer dilution the first time, and primer stock the second time. But if there are degraded, I should still get some absorbance, isn't it?.
This is what the company reply (they were puzzled as well):
"The only expalnation I can give you is that the primer has a very strong secondary structure that traps more EtBr compared to the other primers...maybe this could also be part of the problem why the primer is not working.
If you re-order the same primer again and the new one will work I will raise a credit for you. If you order different primers and these work, then it may be due to the sequence of the primer that was not suitable (might form secondary structures?)."
It makes some sense, although I am not fully convinced.
ok, it is recommended not to use primer dilutions older than 2 months. but i have several primer dilutions older than 2 months that are working well. but still if u have problems with primer stock also, the problem is different i guess.
coming to secondary structure formation, u can test it. there are some programs that we use to check secondary structure formation while designing a primer. i am using IDT oligo analyzer to check hairpin or self-dimer formation. try the link below:
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
if your primer has a strong secondary structure formation, you may not be able to have your PCR product but the primer should still be seen on gel, i think.
the concentration of the primer may be too low (too much lower than what company suggests)which makes the company responsible for the result. u may send the primer back to the company and request a newly synthesized one.
If secondary structures are the issue, there are several workarounds that you can use. First of all, give your reaction a really good denaturation step at the start. Try 93C for 10 minutes (the polymerase will be able to handle that no worries). Sometimes primers can form weak interactions over time, and a good heating will straighten them out.
If that doesn't work, try adding some DMSO or betaine to the reaction.
You should also run a PA gel to see what you have in the primer tube. If you make if a urea gel, you will remove secondary structure; use other primers in adjacent lanes as size markers.