33 replies to this topic

[bouttela]

Hi everyone, Am new to this so any help would be appreciated.  
I have designed primers with restriction enzyme sites at the 5' and 3' ends respectively and am having trouble getting the desired product.   Template is the gene itself gel purified and keep getting bands at approx 100 bp.  I'm thinking that these are primer dimers...Don't want to have to redesign primers.  Another problem is the Tms are high - 68 .  
Thanks,
New frustrated tech

[vgupta]

Hi Boutella,
You can try setting PCR at different annealing temperatures ranging from 60 C to 72 deg. Tm looks okay to me. If it doesn't work, titrate the concentration of Mg2+ from 1.5 to 2.5 mM.
Hope it works out for you.

[bouttela]

Hi vgupta,
Have already tried the annealing temp thing (did two gradients from 50-70).  Thanks though.

[vgupta]

Two gradients, does that mean you tried only at 50 and 70? That is not enough. Try at atleast 4-5 deg difference e.g. 60, 65, 68, 70, 72. If it doesn't work, you could try to use formamide to lower the annealing temperature. You could use vent polymerase, it is a good enzyme. What is the size that you are expecting?

[bouttela]

No I did a gradient from 50 to 70 (50, 53, 55, 58, 60....)   I have tried using Vent (which I normally love) but no luck.  I keep getting huge bands at 50 bp.  The piece I'm expecting is 1.5 kb.    What concentration of formamide?
-bouttela

[vgupta]

Bouttela,
50 bp bands are your primer dimers. For that I did touch down PCR starting from 5 deg above Tm and going down to 2 deg below Tm.
So your cycling conditions would be like 95 2 min, (95 1 min,  73 1min, 68 2 min, 2 cycles), (95 1 min,  70 1 min, 68 2 min, 2 cycles), (95 1 min,  68 1 min, 68 2 min, 2 cycles) (95 1 min,  65 1 min, 68 2 min, 25 cycles), 68 10 min.

Sometimes they just don't go away. You may have to design new primers from a different annealing site. I used 2.5% formamide. did you try titrating Mg2+ from 1.5 to 2.5.

[Niqqie]

I think I might be having the same problem:

My primers Tm are 71.5 and 69.7 oC resp.
I get some small product amplification ~50 bp.
My amplicon should be 220 bp, which is 70-80% GC rich..

So you are saying that I should just try a touch down, starting at 75 down to 68 oC? Without adding formamide or DMSO or Tween or (NH4)2SO4?

The funny thing is that I got this PCR from Zivelin et al:
Improved method for genotyping apolipoprotein E polymorphisms by a PCR-based assay simultaniously utilizing two distinct restriction sites (Clinical Chemistry).

but IT IS NOT WRRRRKN! My PCR reagents and PCR template are working fine though (tested that).

[vgupta]

I think you try PCR with and without formamide and titrate Mg2+ which is the most important thing.

[bouttela]

Niggle,
I think I figured out my problem by using a polymerase specifically designed for GC rich templates ( Thermalace from Invitrogen, but there are others out there too).  I tried the formamide thing but it didn't work.  
Good luck   and Thanks for the help
-bouttela

[vgupta]

I am glad that your problem has been solved. Formamide thing worked for me when used primers with a Tm of 80 deg.

[Niqqie]

For a Mg2+ titration, what concentrations are best to pick? I mean, exactly what values should I be trying?

[vgupta]

Mg2+ at a final concentration of 1.5, 2.0, 2.5 and 3.0 mM should be tried.

[aleruiz]

I'm trying to make more efficient an RT-PCR because, albeit i have my desired PCR product, i wish to get a better reaction. I think the problem is that the primers are getting in dimers. I've heard about the use of detergents or DMSO in the reaction mix but i'm not sure about this. Does anyone knows a way to do it without change the Tm?

Aleruiz

[tfitzwater]

Dimers tend to form when the template concentration is low.  If you can, try titrating in more of the template.

Adding yeast tRNA carrier can reduce the amount of template that sticks to the tube walls, making more of it available for amplification.  

When designing your primers, check them for homo-dimer and hetero-dimer formation with the Oligo Analyzer program at the IDT website www.idtdna.com.

[ocean]

dmso can be used between 2-10%, but the higher you go the  less active the polymerase becomes so you may no get the same amount of cylces.   u can try at 2 and 5% to be safe.