What do you mean by melting temperature??

Normally, no because at 59C, an oligo with a Tm of 40C will not anneal to the template. I say normally because anything can happen :-)

Barry

Hi, the primers given in the attached paper (Primers SCTOX5–1 (5′-GTCTATACTCGACAATAGTCC-3′) and SCTOX5–4 (5′-GTCCTTCTGAGAGAACACTA-3′) have Tm 51,9°C and 54,4°C. Or do you mean the RAPDs?

Attached a paper (RAPD)!

Cheers, Nadine

Hi I guess there is a small typo error or misunderstanding of what you have mentioned up there in your question regarding primer annealing and melting temp. As of my knowledge the melting temp should be higher than the anneal temp unless and other you have got a superman primer. In case if i was wrong the best choice for this situation is doing a gradient PCR to identify the optimal temperature for you primers. Beforehand you can check the best temperatures using Perlprimer and primercheck (free softwares) or some other paid softwares Lasergene and etc. Hope this helps a bit

Cheers

Aditya

Many thanks friends ...

The situation is ... I am trying to amplify Tri5 gene in stachybotrys charataum . I used the attached paper protocol. (Primers SCTOX5–1 (5′-GTCTATACTCGACAATAGTCC-3′) and SCTOX5–4 (5′-GTCCTTCTGAGAGAACACTA-3′) ... According to the paper the protocol was an initial denaturation

for 90 s at 93°C, followed by 30 cycles of 20 s at

93°C, 30 s at 59°C, and 60 s at 72°C.

I did not get any bands .....

I return to the information of primers in company sheet I discovered that both primers with a Tm of 40C.... So in my mind it could not be anneal at higher temperature (59) as mentioned in the paper. ...

I run gradient PCR from 60 to 36 C also no bands .....

I used each primer as RAPD primer at 36C with my samples. Both primers gave many bands !!!!!.

So is there any explanation !!!!!!!!!!!!!!!

Hi Youssuf, read my first response! I calculated the Tm with this program: http://www.iit-biotech.de/iit-cgi/oligo-tm.pl

The average Tm of both primers are ~53°C. 59°C is very high for annealing (without any information (given PCR protocol) I would start with an annealing of 50°C).

That you didn't get any bands might have several reason:

DNA too high/ too low

MgCl (or MgSO4) concentration is not optimal

Try HotStart Polymerase instead of "normal" Taq

DNA contains inhibitors or is not clean

All the best, Nadine

Dear Nadine

Every thing is optimized because I got nice amplification with Betatubulin gene and ITS regions. I used hotsart taq and I have very good master mix as I told you this condition were quite nice for other gene amplification !!!! I run gradient with thosr primers from 36 to 60 and no bands !!!

Thank you so much for thinking with me

Youssuf

Hi Youssuf,

for me the situation looks like these primers does not have amplification site on your DNA

This can happen for several probabilities like;

1- maybe there is some degradation happened in your DNA, you can run the DNA on a gel to check for this.

2- maybe there is a mutation , so this gene's deleted

I hope you can find another primer set for the same gene :)

good luck

Hi Youssef

This is another exemple of the variability of the methods for calculating PCR primers. You should retain programs using enthalpy, not suppliers Tm estimations which are often rough estimations. But Tm depends on primers sequence and also concentration as well as Mg2+ and salt in in PCR mix . I would recommend this web page from ABI "http://www6.appliedbiosystems.com/support/techtools/calc/"

which take this in account.

It is a good point to have a control pCR to check if DNA samples are OK. But there is still a possibility that they might be some differences between PCR: ie ITS amplification often works better as these regions are often in several copies. In addition (as mentioned Nadia) , you may have SNPs in your target sequence.

Temperature range is also Primers dependent: you will get good amplification on a wide temperature range with some primers whereas for some others you will not get anything at too low or high temperature .

But before anything I suppose that your target sequence is in Genebank : did you check that the primers match the sequence at 100% : there might have been a typo error or even worse they might be on the same strand (it happened to me once).

Finally I would also mention that some Taq are more processive than others ie: Extaq f Takara is really efficient so are Kappa or fusion Taq.

Good luck!.

JL

Hi Nadia

If my primers does not have amplification site on your DNA. What about the many bands I got them when I used my primers separately as RAPD primers!!!!!

I already ordered new set of primers

Many thanks

Youssuf

Hi Jean-Luc

Yes I checked that the primers match the sequence at 100%. I run blast with those primers against genebank and I found them in the complete sequence of this gene in this fungus. !!!!

Many thanks

Youssuf

About many bands at low temperature, this is very likely unspecific annealing (like for RAPD).

But did you check the presence of primers dimers: they might completely inhibit your PCR, especially if you are at a lower temperature, and this may explain why they used a so high annealing temperature in the reference paper.

You can lower the primer concentration in the mix (up to 0.4mM), use a high Tm (same as paper), and try to add DMSO up to 5 percent of the reaction volume.

Good luck.

JL

I can not see any primer dimers in the gel. Thank you so much for your suggestions, Sure I will try them

Many thanks

Youssuf

GC content of the product might be to high that it was not able to generate further products, so DMSO, BSA, Glycerol may be useful in this case. another possibility, primer have stem loop structure which prevent binding of primer to template.