PCR Primers same as cDNA Primer?

[galgur]

Hello,

When i run a PCR I normally isolate RNA first then RT to cDNA and run PCR with that. This time i isolated the genomic DNA from the sample and ran PCR using the beta-actin primer i always used. Dimers were faintly visible on my gel but nothing really came up like it normally would have or should have. I was told that when running PCR with genomic DNA, a different kind of primer had to be used; one that is specific to genomic DNA. I'm confused as to why it would matter or if it matters at all.

[Kellee]

Primers are different for cDNA - they are polyT or random primers whereas pcr primers are specific primers generated from your DNA template sequence.

Primer usage depends on your definition of primer types, and how you originally designed your primers. If the primers you use on your cDNA were designed to cover exon/intron boundaries, then those primers are not going to work right on genomic DNA because the introns are present. In that case you would need to design new primers. These will be very similar in that they'll be 20-ish-mers of DNA, but will have a different sequence.

Since all you're seeing is primer-dimers, I'm guessing this is the case. You could try messing around with the annealing temperature and MgCl2 concentration though if you think it should be working, and compare the sequences of the primers, cDNA, and genome to make sure they're all lining up and matching.

[sravani_june21]

why the c dna contain the hair pin like structure, how it is act as primer?

[camille]

Hi !!

I want to check if 4 genes are organized in operon. what primers i must choice after rna extraction to convert rna to cdna ???

[Xandra]

I want to perform revers transcription with random hexamer primers and I have lyophilised Primer random p(dN)6 from Roche (ref. 11 034 731 001), 50 A260 units.Please, who can help me to find out how to re suspend these primers to have 1 micro gram per micro liter solution?
Many thanks!

[Aga]

Originally Posted by Xandra

I want to perform revers transcription with random hexamer primers and I have lyophilised Primer random p(dN)6 from Roche (ref. 11 034 731 001), 50 A260 units.Please, who can help me to find out how to re suspend these primers to have 1 micro gram per micro liter solution?
Many thanks!

What do you mean by units? 50 units - what specifically does it mean? Is it 50μg/ml? It corresponds to optical density (OD) = 1.

[Xandra]

Hi Aga, Hi everybody!
This is my problem, I do not know the mining of that "50 A260 units", this is written on the primer vial, no micrograms, nothing else, that is why I have been confused.


Thanks!

[Aga]

I found this:

1 A 260 unit of double-stranded DNA = 50 µg/ml
1 A 260 unit of single-stranded DNA = 33 µg/ml
1 A 260 unit of single-stranded RNA = 40 µg/ml

Source: [Only registered users see links. ]

[Xandra]

Thanks!