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oak945's Content

There have been 4 items by oak945 (Search limited from 25-September 19)

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#132458 Help with primer design through PRIMER3

Posted by oak945 on 06 April 2012 - 07:29 AM in PCR, RT-PCR and Real-Time PCR

I used Primer blast, its giving well designed primers but for few of my target genes have very small exons flanked by large number of intron.
After cross-checking with UCSC PCR tool, its coming out to be not ok from exon junction or intron spanning primers.

#132252 Help with primer design through PRIMER3

Posted by oak945 on 03 April 2012 - 03:29 AM in PCR, RT-PCR and Real-Time PCR

Hey Quasimondo
Thanks for our suggestion, its working well.
I have ordered the primers and lets see how well its working

#132241 Help with primer design through PRIMER3

Posted by oak945 on 02 April 2012 - 09:57 PM in PCR, RT-PCR and Real-Time PCR

Hello there,
How I can design a intron spanning primer (forward primer on 1st exon reverse primer on another exon) or exon junction primer (primer spanning exon exon junction) through Primer 3.

Thanks in advance for help.

#127303 Same non specific band for different target proteins

Posted by oak945 on 18 January 2012 - 11:10 PM in SDS-PAGE and Western Blotting

Hello there,
I have to quantify 3 different target proteins. but each time I do wetern blot, I get a very distinct and sharp nonspecific band above 100kD.
I have tried:
1. different chemicals form different vendors,
2. Primary antibodies obviously different
3. blocking both with BSA or milk
4. vectastatin secondary antibody (two anti rabbit, one anti mouse), avidin biotin complex-HRP conjugated (this is common for all runs)
5. developed with DAB (common for all runs)
6. even tried different tissues like brain tissue, retina, spinal cord
7. INTERSTING is that band appears same intense in all my 9 groups that is independent of total protein concentration (as ihave tried loading total protein with large differences).

What could be your opinion?

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