6 replies to this topic

[hebegebe]

Hi, I wanted to continue with this thread:

http://www.protocol-online.org/biology-for...osts/40260.html

but couldn't figure out how.

In it, pcrman said:

"We order two oligos, one 15mer (T)15, and another 18mer (T)18. The oligos are just Ts, no overhangs of any kind. When the oligos arrive, just mix them in equal amount to make oligo (dT)15-18 primers with a final concentration of 0.5 ug/ul. It works equally well as oligo(dT) primers we buy from companies. The cost is only a fraction of that."


So here's my question:

Do the primers need to be hplc grade, or is 'standard' grade ok? I.e. is RNAse contamination in standardly produced primers going to be an issue during cDNA synthesis? (Info from idtdna.com suggest they might be? http://eu.idtdna.com...e.aspx?id=13136 )

Cheers!

[phage434]

Standard grade oligos should work fine. I see no reason to suspect RNAse contamination, unless you add it.
This is a very cheap experiment to try. We use TTTTTTTTTTTTTTTTTTV oligos, which bind at the first non-A base position in the mRNA.

[pcrman]

We ordered the oligos as desalted to make our own oligo dT primer and have never had RNase contamination problem.

[hebegebe]

I appreciate your replies

I have a few further questions for takers:

Do you phosphorylate the 5' end? It increases the cost I was quoted approximately x3.

Why do some manufacturers supply a mix of different length oligo dT e.g. 12-18 dT - why not just 18 dT? I assume it increases the range of temperatures the primers will work at.

Anchoring:
1) Is it important - pcrman doesn't appear to bother, while phage434 does. Perhaps it depends on the cell type under study (I'm using human cells).

2) 1 or 2 nucleotide anchor? Is there a benefit in adding an N after the V? i.e. TTTTTTTTTTTTTTTTTTVN . Is it just to increase the Tm?


Cheers again!

Edited by hebegebe, 07 October 2009 - 11:29 PM.

[stardust]

Hi,

what does V stand for?

Stardust

[phage434]

B = not A (any of G, C, T)
D = not C
H = not G
V = not T (U was already used for uracil)

[gleb.kudr]

Adding N will decrease your actual primer concentration four times. It isn't worth it.