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Tagged primers and a second PCR targetting the tags

Primers

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5 replies to this topic

#1 GradMicro

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Posted 18 December 2012 - 01:42 PM

I need to add a barcode to my multiplex PCR products. What I plan to do is to add an extra 18 nt sequence to all the primers of my multiplex (both reverse and forward), purify products after PCR.
Develop another primer that has the sequence of the 18nt added to the primers of the multiplex. Add the different variants of barcodes to this second primer and use the purified product from step one as the template to run.
If I add the same sequence in the 5 ' to 3' direction of both forward and reverse primers in step one, can I use a single primer as both the forward and reverse in step 2? I know this is a very naive question and sorry for that, I get mixed up with the sequenes of complementary, and reverse complimentary sequences etc.

#2 pcrman

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Posted 18 December 2012 - 04:20 PM

I think it is doable to amplify the product with a single primer and believe it works the same way as two primers.

#3 phage434

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Posted 18 December 2012 - 06:21 PM

Yes, this works. Use twice as much of the single primer.

#4 GradMicro

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Posted 10 January 2013 - 01:23 AM

Thank you very much for the replies and sorry for not acknowledging earlier.
Unfortunately, I can not seem to be able to optimise the second PCR, tried all possible things such as tep gradients, primer concentration gradients, gradients of other contituents, PCR additives.
The bands are there, but not sharp enough.
Would it be due to the use of same primer? Posted Image

#5 phage434

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Posted 10 January 2013 - 05:52 PM

I think that is unlikely. More common would be a poorly designed primer. Many years ago I decided that optimizing pcr reactions was no fun, and that redesigning primers would usually solve problems much faster than titrating magic pcr additives.

#6 GradMicro

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Posted 14 January 2013 - 07:14 PM

Something odd is happening with my PCR
1. For the second PCR, as primers I ordered both the sequence of the original tag and the antisense of the original tag as primers. When the second PCR is done with the primer with the antisense sequence of the original tag, bands are very weak. However, when it is done with the primer with the seqence that is the same as the original tag, bands are strong.
2. I have two sets of primers for the first PCR, one which is the tagged primers, and second primers without tags. Even the product of the first PCR done with primers with no tags are amplified by the second PCR. I thought this is due to the presence of excess primers from the first reaction itself and tested again with purified products of the first PCR. Still, band present. As the tagged varient of the second set of primers have not binding sites in the product of the PCR,the only explanation I can think of is that it is just the product of the first PCR, which even when diluted is concetrated enough to give a band in the gel.
I have not done nested PCR or this step out PCR before, I would like to know if this is a known phenomena.





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