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No Amplification -- Primer Dimer

No Amplification -- Primer Dimer - PCR - Polymerase Chain Reaction Forum

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  #1  
Old 08-30-2011, 05:08 AM
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Question No Amplification -- Primer Dimer



Hello,

I'm trying to amplify a gene from Z. mobilis genome. As I tried I'm not getting any amplified product. And also a concentrated primer dimer. The product is 1.6KB. Will the amplified product vanish because of dimer dominance? What's the trouble? How to rectify??
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Old 09-02-2011, 12:13 PM
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Default Re: No Amplification -- Primer Dimer

If possible post the gel picture with your question. that will help to answer your question.

Concentrated primer dimer means bad primer design (check your primer how strong can your primer can anneal each other either itself or with other primer). And if you are doing for first do a gradient pcr, and reduce your primer concentration.
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Old 09-05-2011, 08:32 AM
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Exclamation Re: No Amplification -- Primer Dimer

Thanks for the response sir. As you suggested sir, my primer is long and forms loop. But the thing is that I have to add restriction sequence and ribosome binding region sequence along the primer. It was unavoidable. Can the increase in annealing temperature help in working this out??? Primer conc. is 100ng/ul. Should it be diluted further??? Gradient Pcr helps in finding the apt annealing temp??

Note: I'll try to post the gel snap soon sir.
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Old 09-05-2011, 08:52 AM
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Default Re: No Amplification -- Primer Dimer

Thanks for the response Sir. As you assumed my primer is long and designed in a way that it has restriction site and ribosome binding region sequence. It forms a hairpin too. But its unavoidable. Can the annealing temperature increase help in rectifying this? My primer's working conc is 100ng/ul. Should this be diluted even more? And will the gradient pcr helps in finding the best annealing temp?

Note: I couldn't post the gel snap. Sorry Sir
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Old 09-05-2011, 11:02 AM
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Default Re: No Amplification -- Primer Dimer

If you are not able to control the Primer design. Then you have to standardize your experiment with annealing temperature as i suggested earlier do a gradient PCR.

And regarding primer concentration was it 100ng/L or 100ÁM.

The recommended primer concentration for PCR is between 0.1μM and 1μM of each primer
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Old 09-06-2011, 03:35 AM
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Default Re: No Amplification -- Primer Dimer

Primer was supplied as 167ug and I suspended it in 167ul. Then I further took 10ul and made it as 100ul. So the final conc was 100ng/ul. What is to be done?? I dont get the concept of primer dilution. Sorry Sir if its a stupid question
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Old 09-06-2011, 06:53 AM
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Default Re: No Amplification -- Primer Dimer

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Old 09-06-2011, 08:41 PM
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Default Re: No Amplification -- Primer Dimer

I think you are using too much primer. Try to reverse calculate how much μM of primer do you used for your reaction (you can calculate it from MW of your primer)
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Old 09-08-2011, 03:41 AM
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Default Re: No Amplification -- Primer Dimer

Ok Sir. Will try the calculation manually. Thanks for guiding sir..
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Old 09-09-2011, 06:09 AM
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Default Re: No Amplification -- Primer Dimer

Concentration (Volume 1ml) 14.2pmol/ul
Volume for 100pmol/ul -- 142ul

So the dilution of primer should be done in 1.42ml then it yields 10pm/ul. Is this calculation right sir?
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