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pcrmossad's Content

There have been 5 items by pcrmossad (Search limited from 21-September 19)


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#67469 RT-PCR standard curve dilutions

Posted by pcrmossad on 20 April 2010 - 12:50 PM in PCR, RT-PCR and Real-Time PCR

You should always have standards that span the area where your samples are. So if you except samples to be maximum 10 fold downregulated, you don't need to do 0.01 dilution in your standard curve.
Usualy more wider dilutions are used because they span all possible concentrations, but if diferent dilutions have different amplification efficiency (due to inhibition for example) it may skew the slope of the standard curve. In that case for concentrations around 0.5 - 0.25 the standard curve with 0.5 and 0.25 dilutions could be more accurate. It really depends on the range of your samples.


thanks again !



#67315 RT-PCR standard curve dilutions

Posted by pcrmossad on 19 April 2010 - 01:29 PM in PCR, RT-PCR and Real-Time PCR

Hi all,

I am using RT-PCR for validating microarray data for some time now. usually I use 1, 0.1, and 0.01 dilutions to generate my standard curve. in rare cases I have used 0.2 .

my question is should I always use these dilutions? cant I use 1, 0.5, 0.25 etc dilutions to generate a standard curve? what are the disadvantages of using this kind of dilutions apart from wasting control samples?

thanks in advance.



#60719 Designing primers with ESTs

Posted by pcrmossad on 28 February 2010 - 12:41 AM in PCR, RT-PCR and Real-Time PCR

it is totally feaisble to design primers based on EST sequences if there is no known mRNA sequence and to cite the sequence in your papers. The more EST sequence alignment for the region of your interest, the safer that the region is a true mRNA sequence.


Thanks for the reply. appreciated.



#60710 Probe manufacturers, who makes best probes?

Posted by pcrmossad on 27 February 2010 - 04:59 PM in PCR, RT-PCR and Real-Time PCR

I have gotten some FAM/BHQ probes from a vendor and although they work initially, they go off quickly (within weeks.) The primers (from another source) seem fine.

I do not freeze thaw repeatedly, I protect from light, and I keep them stored at -80 long term. I am thinking the synthesis is not good. It has been a problem with all the probes we have ordered from this company.

Where do you get your real-time PCR probes, and have they been stable?



I buy them from thermo electron. If you aliquote them and store them at -20 , theiy are fairly stable, and even worked after one year.

http://www.thermo.co...ome/1,,,00.html



#60709 Designing primers with ESTs

Posted by pcrmossad on 27 February 2010 - 04:52 PM in PCR, RT-PCR and Real-Time PCR

Dear all,

I have decided to design primers using EST sequences since the species (sus scrofa) I am involved with have less mRNA data in Genebank. I am doing RT-PCR for my microarray validation. What are the pit falls and risks of designing primers using ESTs. Can I cite them like mRNA sequences? Please let me know what you all think. thanks in advance.




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