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High Tm on primers - cannot get a product


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#1 Knights

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Posted 16 August 2010 - 11:54 AM

Hey guys,

I've been having a hard time amplifying a product that's CG rich. The primers are CG rich, with the Fw and Rv primers having a Tm of 70C (using the A/T=2C and G/C=4C method). The Fw primer has only 3 A/T! The method we use to find the annealing temperature has worked without any issues in this lab, after I tried this PCR reaction I tried to amplify a different product and had no problems there so I know it's not my reagents or my temperature calculating methods.

So needless to say, I'm stuck. I've been trying different things all week long. I've used different templates, played with the temperature, etc. and cannot get a SINGLE band. The product is about 900bp long. I have also tried old primers we had for the same product (just a little bit further up and downstream), with no success. The previous student did not have a good record keeping habit so I cannot even see if they were originally successful.

Any suggestions? I've never done a 2step-PCR. I tried that and I don't know if it failed or I just didn't set it up correctly.

Here's what I've tried/used:
Phusion
Their CG buffer + MgCl2
Annealing temperature ranging from 58-66C

Thanks in advance for any advice.

#2 Micro

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Posted 16 August 2010 - 03:11 PM

Just to clarify are you using Finzymes' Phusion DNA polymnerase? If yes, they have a Tm calculator on their website that is specific for their enzymes and I've found 2-step is often required in high GC for this brand of products.
Also, when I've used this brand of products I get excellent quality products for about half to three-quarter of the tube of DNA polymerase and then the product quality nose dives rapidly. If you are using an old tube you may want to double check the enzyme is still functioning properly.

#3 phage434

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Posted 16 August 2010 - 06:05 PM

Usually adding 5% of a 1M Betaine solution solves GC rich pcr problems. You can control the primer Tm by simply making it shorter (though it will be less selective). Check the primers for homo and heterodimers and for hairpin formation (with the 3' end bound, leaving a 5' overhang). The IDT web site has a tool for analyzing these issues.

#4 Adrian K

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Posted 16 August 2010 - 06:13 PM

Depends on how you want to get the band of interest.
For a GC rich amplicon, first you must make sure that your primers are 17-22 bases long... I understand that the annealing temperature are high, but with a shorter primer I always cant get anything.

I used to deal with this situation before. Here are my suggestion:
Try lower your annealing temperature for 10C (~50C) for 30s, extension temperature 72C for 40s.
You should expect to see multiple bands here. Gel excise and purify the band which is ~900bp long.

From here, you can try to PCR the purified band. If the PCR doesn't work, do a sample pooling.

This is how I used to get rid of those hard template, the hard way.

Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#5 Knights

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Posted 17 August 2010 - 05:18 AM

I've tried Finn's website Tm calculator. That's how I tried doing the two step.
I already checked my primers too.
Thanks for the annealing temperature decrease and band isolation. I'll try that today.
Any other ideas would be great just in case.

#6 Adrian K

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Posted 17 August 2010 - 08:42 AM

Keep us update about your outcome. All the best. Good luck.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#7 Knights

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Posted 18 August 2010 - 05:46 AM

Adrian, I got a [faint] band! It's the first time this week I've been able to get SOMETHING. It's faint and weak and I'll have to now work on optimizing it, but it's there and it's the right size. All I did (so far) was drop the temperature to 50C. I tried using both my genomic DNA and vector that has the gene, and the vector is the one that gave me the band.

This is exciting and I would have honestly not thought about decreasing the temperature that much.

Thank you so much for the insight! Let's hope I can get a nice band today.

#8 HomeBrew

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Posted 18 August 2010 - 06:06 AM

If you have the gene in a vector, can't you just pop it out with a restriction digest?

#9 Adrian K

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Posted 18 August 2010 - 09:39 AM

If you have the gene in a vector, can't you just pop it out with a restriction digest?


I do agree with HomeBrew, if is in vector, why not try with a restriction digest?

Adrian, I got a [faint] band! It's the first time this week I've been able to get SOMETHING. It's faint and weak and I'll have to now work on optimizing it, but it's there and it's the right size. All I did (so far) was drop the temperature to 50C. I tried using both my genomic DNA and vector that has the gene, and the vector is the one that gave me the band.

This is exciting and I would have honestly not thought about decreasing the temperature that much.

Thank you so much for the insight! Let's hope I can get a nice band today.

You are welcome. Try lower it down even further and see how it goes... we won't know until we tried it.
Also, as phage434 said, add 5% of a 1M Betaine solution to solves GC rich pcr problems. For myself I tried 5% DMSO... just to let you know with DMSO I experience some slight different annealing temperature profile.
You can also do a temperature touchdown PCR, say, 50C for 10 cycles, and gradually reduce to 45C....etc... however, it really need lots of trial and failure.

p/s: just curious, what are you trying to do actually?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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