5 replies to this topic

[Ivan]

I am trying to design a tagged RT primer for detection of the negative strand of a certain RNA virus.  However I do not find any basic rules for primer design of tagged primers.

Now I just use the regular primer design rules and make sure they amply to both primer and tag.  

Does anyone have any suggestions, since multiple try outs have failed with my tagged primer; while the normal primer worked.

[gleb.kudr]

Ivan, on Sep 14 2009, 08:05 AM, said:

I am trying to design a tagged RT primer for detection of the negative strand of a certain RNA virus.  However I do not find any basic rules for primer design of tagged primers.

Now I just use the regular primer design rules and make sure they amply to both primer and tag.  

Does anyone have any suggestions, since multiple try outs have failed with my tagged primer; while the normal primer worked.

The basic rule is that your tag annealing temperature must be lower than the other primer (to prevent it from unwanted interaction with DNA).

Edited by gleb.kudr, 17 September 2009 - 03:31 PM.

[Ivan]

gleb.kudr, on Sep 18 2009, 01:31 AM, said:

The basic rule is that your tag annealing temperature must be lower than the other primer (to prevent it from unwanted interaction with DNA).

Indeed sounds logic, didn't check for that.  Thanks!!!

Edited by Ivan, 18 September 2009 - 03:48 AM.

[Ivan]

gleb.kudr, on Sep 18 2009, 01:31 AM, said:

The basic rule is that your tag annealing temperature must be lower than the other primer (to prevent it from unwanted interaction with DNA).

a difference of at least 5°C should be okay I guess? With the RT priming above the annaeling of the TAG

Edited by Ivan, 18 September 2009 - 03:49 AM.

[gleb.kudr]

Ivan, on Sep 18 2009, 01:01 AM, said:

gleb.kudr, on Sep 18 2009, 01:31 AM, said:

The basic rule is that your tag annealing temperature must be lower than the other primer (to prevent it from unwanted interaction with DNA).

a difference of at least 5°C should be okay I guess?

Okау but to make calculations you should look only to GC content/ratio of each part to discard it length impact (basically > length > t anneal. but it is useless in this case).

Edited by gleb.kudr, 18 September 2009 - 04:43 AM.

[Ivan]

gleb.kudr, on Sep 18 2009, 02:39 PM, said:

Okау but to make calculations you should look only to GC content/ratio of each part to discard it length impact (basically > length > t anneal. but it is useless in this case).

I have a new tag with low GC ratio especially at the 3’ end.  I looked at my former tag and the GC content was comparable to the primers GC content, so fingers crossed and hope the improved version does his trick.