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sri lankan

Member Since 06 Sep 2009
Offline Last Active Feb 15 2013 06:32 PM

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15 Sep 2009 - 00:21
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Topics I've Started

PCR product purification

14 February 2013 - 08:16 PM

I need to PCR products that range from 200 - 600 bp. The kit used is QIAquick. i follow the default protocol with no modification. PCR volume is 50 ul, I use 5 ul to run a gel so I use 45 ul for purifcation. Bands for all PCRs are very shart/ intense. However, after purification the maximum concentration (by nano drop) that I can obtain is around 30ng/ul. Elution volume 50 ul.I need to acheive a concentration above 50ng/ul. Reducing elution volume ( i use molecular grade water) is not very practical for me as I need to obtain about 2.5 ug of DNA (thus conc needs to be > 50 ng/ul and volule 50 ul).

What I plan to do
Incubate for a longer time prior to elution
Centrifuge for a longer time.

Any other suggestion are most welcome.

Thanks a lot.

Tagged primers and a second PCR targetting the tags

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.

Barcode generator for PCR primers

13 December 2012 - 08:11 PM

I am looking to tag a set of primers with barcodes for checking the possibility of adding an additional multiplexing step in massive parallel sequencing.
I would like to know if these is an open source software that can be understood by a total novice, which would generate PCR primer compatible barcodes? The primers are for a normal PCR.

Thank you.

Barcoding PCR products

11 December 2012 - 12:25 AM

I need to barcode PCR products before sequencing. However, as my PCR is a multiplex, barcoding the primers are not an option. Is there a possibility to generate short DNA barcodes (4 - 6 nt) and use a template independant ligase to add it to the PCR product?

Transcription of PCR products without a primer coupled with promotor sequence

02 December 2012 - 07:03 PM

Hello,

Is there a way to transcribe PCR products wihtout using a primer that has a transcription promotor. I plan to do base specific cleavage followed by MALDI TOF for strain differentiation. My PCR is a multiplex PCR with 15 primer pairs. Thus, tagging the primers is not a possibility.
Any alternatives are most welcome.

Thank you

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