ladder for acrylamide microsat work -- need 2bp interval - ladder for acrylamide microsat work -- need 2bp interval (Aug/10/2006 )
hi guys
just wondering if such a product exists commercially: a ladder with very small (i.e. 2bp) intervals??
if so -- could you give me the company name? (I've already had a look througth the sigma calendar but maybe I'm missing something obvious)
--I'm doing microsatelllite scoring on polyacrylamide gels - - and I need to be able to discriminate and catalogue these tiny differences - regular ladders are obviously not on a fine enough scale and I don't have old positives for which we already know the sizes since I'm starting work with new primers
any help/input could give would be awesome!!
thanks!!
just wondering if such a product exists commercially: a ladder with very small (i.e. 2bp) intervals??
if so -- could you give me the company name? (I've already had a look througth the sigma calendar but maybe I'm missing something obvious)
--I'm doing microsatelllite scoring on polyacrylamide gels - - and I need to be able to discriminate and catalogue these tiny differences - regular ladders are obviously not on a fine enough scale and I don't have old positives for which we already know the sizes since I'm starting work with new primers
any help/input could give would be awesome!!
thanks!!
I know only the ultra low range ladders from NEB & Fermentas, they have 25 bp steps. Less seems not useful, as you will not see very much except a smear. For microsats you need some bands as standard in the size range you have, the rest can be calculated/estimated by software.
hi
yeah -- I saw those low range ladders too
--but the thing is something with a smaller than 25bp interval would be fine because with the size of ancrylamide gel and the length of time we run it, it ensures that we get really good separations bewteen bands that are only 2bp apart
--normally we do use a known standard in our range -- but the problem here is that since this is a whole new primer -- I don't have a known standard in the right range
thanks for your response though -- I appreciate it and any more input you might have ;o)
yeah -- I saw those low range ladders too
--but the thing is something with a smaller than 25bp interval would be fine because with the size of ancrylamide gel and the length of time we run it, it ensures that we get really good separations bewteen bands that are only 2bp apart
--normally we do use a known standard in our range -- but the problem here is that since this is a whole new primer -- I don't have a known standard in the right range
thanks for your response though -- I appreciate it and any more input you might have ;o)
Perhaps constructing your own ladder e.g. with lambda and RE's, similar to lambda PstI, but its time consuming. I did it some time ago with Vector Nti (or NEBCutter) and my sequence to get fragments of appropriate size. It shows you fragment numbers, sizes, cleavage sites etc etc., realtively comfortable if you make multiple digests.
thanks again!
I think I might have to try that out!
)
(that was supposed to be a smiley face)
Fermentas step ladders work as claimed. You just have to (a) use the conditions shown on their pictures, and (
use high quality reagents for making the gel to avoid smearing.
The best they do is bp increments:
http://www.fermentas.com/catalog/electroph...erulers.htm#5bp