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Multiplex PCR


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12 replies to this topic

#1 susanna

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Posted 07 September 2011 - 01:42 AM

Hi,

I did an multiplex PCR experminent with two primersets.
I first tested both primerpairs individual, and they are very specific, but when i add them together, they also amplify other products besides the two products i designed them for.
I have to admit that the FP of the first primerpair overlaps one nucleotide of the RP of the second primerpair. Should this be an issue? So not, what could me the problem and how to solve this?

greetz

Susan

#2 hobglobin

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Posted 07 September 2011 - 07:03 AM

Primer dimers can be a problem, especially if they are stable. You can use software as the usual primer design programs to find out if the primers don't fit good (primer 3, perlprimer, netprimer) or this software, that is not bad.
To avoid this you may design new primers that do not form dimers and/or other structures you don't want, you can try to add additives that sometimes help (BSA, betaine, etc), and a hot-start often helps, too.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#3 Adrian K

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Posted 07 September 2011 - 08:33 AM

Usually, using a higher temperature will solve the problem. Try add DMSO 5%. Possible to show your gel here?
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

#4 Ameya P

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Posted 07 September 2011 - 10:20 PM

Along with Adrian's high temperature, if possible, slightly reduce the concentration of the primers you are using for the multiplex.

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#5 susanna

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Posted 08 September 2011 - 01:59 AM

HI,

I didnt run them on gel, i saw the aspecific product on qPCR.

I'll try to sent the figure.

greetz

#6 susanna

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Posted 08 September 2011 - 02:14 AM

i just can not find out how to past/import my figure

#7 Adrian K

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Posted 08 September 2011 - 10:12 AM

try to "print screen", paste and edit in "paint", save and upload to "imageshack", finally put the link here?
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

#8 susanna

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Posted 08 September 2011 - 11:42 PM

here he is:

http://my.imageshack.us/v_images.php

the red curve (highest one) is the aspecific product

Edited by susanna, 08 September 2011 - 11:43 PM.


#9 Adrian K

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Posted 09 September 2011 - 09:54 AM

I'm sorry, I dont think you had put the correct link...
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

#10 hobglobin

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Posted 09 September 2011 - 10:40 AM

you can also post it as attachment (hidden in more reply options) here

Edited by hobglobin, 09 September 2011 - 10:44 AM.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#11 susanna

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Posted 12 September 2011 - 12:07 AM

Hi,

here's a presentation of the problem with the aspecific product,

greetz

Attached Files



#12 Adrian K

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Posted 12 September 2011 - 12:21 AM

I'm not expert in Real-time PCR, however, I do find that your CT value is quite high, >30
I can see there are some possible primer dimmers in your melting curve in your last slide: is this what you mean by "amplify other products"?
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

#13 susanna

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Posted 15 September 2011 - 05:52 AM

yes, but the strange thing is that if you use the primerpair seperated you dont see this product. Probably primer heterodimer between the 2 primerpair, like you say




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