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questions about pcr products after pooling


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#1 claritylight

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Posted 24 January 2010 - 09:53 AM

please confirm in the different scenarios. i am basically wondering how pooling various pcr products will affect the originals.

1) i have 3 amplicons after PCR, 10ul pcr volume. all show a band at 500bp when run on a gel. i pool all 3 amplicons together to make a total 30ul volume. when i run some of this pool on one lane of a gel again, will i just see one large thick band at 500bp or some kind of smear or what?
2) again, i have another 3 amplicons after PCR, 10ul PCR volume. amplicon #1 is 200bp, #2 is 400bp, and #3 is 500bp. I pool all three amplicons together to make 30ul total volume again. when i run some on one lane of a gel again, i will see 3 bands at 200bp, 400bp, and 500bp? or will i see a smear or some bands in the middle of 200bp-500bp?

#2 swanny

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Posted 24 January 2010 - 09:20 PM

This sounds suspiciously like a homework question.... Like, why would anyone do 10ul reactions and bother pooling them?

What do YOU think will happen?
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#3 claritylight

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Posted 24 January 2010 - 10:45 PM

This sounds suspiciously like a homework question.... Like, why would anyone do 10ul reactions and bother pooling them?

What do YOU think will happen?


I'm sorry if you think it is a homework question since it is not. I ALREADY gave what I thought in my questions as doubts and said I would like to have those doubts confirmed. 10ul reaction volume was an example volume, so what exactly is the problem there? I actually don't know what volume is usually used for pooling PCR products, perhaps another question someone can answers.

If you cannot answer the questions I have, I would appreciate it if you would at least stop your judgements. Thanks.

#4 stylothecancer

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Posted 25 January 2010 - 01:25 PM

please confirm in the different scenarios. i am basically wondering how pooling various pcr products will affect the originals.

1) i have 3 amplicons after PCR, 10ul pcr volume. all show a band at 500bp when run on a gel. i pool all 3 amplicons together to make a total 30ul volume. when i run some of this pool on one lane of a gel again, will i just see one large thick band at 500bp or some kind of smear or what?
2) again, i have another 3 amplicons after PCR, 10ul PCR volume. amplicon #1 is 200bp, #2 is 400bp, and #3 is 500bp. I pool all three amplicons together to make 30ul total volume again. when i run some on one lane of a gel again, i will see 3 bands at 200bp, 400bp, and 500bp? or will i see a smear or some bands in the middle of 200bp-500bp?



Hi claritylight,

for question 1, in my experience, i will get a bankd of 500 bp after i pooled the sample. however, sometime i do get some smear, which i do not know why.

for question 2, i will be band 200, 400 and 500 bp as well.

however, if the band is very faint in your original 10 uL tube, sometimes you won't see anything after you pool your sample. probably due to dilution effect, I presume. afterall, this is my experience.

hope this would help ......




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