Rf: Plasmid sequencing: Methods to improve resolution

LOGAND logand at msdos.ensam.inra.fr
Thu Jul 6 07:54:17 EST 1995


Frankie, T. C. Lee wrote:

>I would like to know how I can get better-resolution sequencing (bands 
>towards the end of the gel are hardly distinguished). Moreover, are there 
>any methods to increase the number of sequence obtained each time(other than 
>buffer-gradient page)?

>Information about my sequencing procedures:

>I am now using T-7 sequencing kit with S35 dATP label. I have cloned my PCR 
>product into PCR II vector before performing sequencing.

I posted a similar question some weeks ago asking for info on using wedge 
spacers - this is one technique you can use. Wedge spacers (thick at bottom of 
gel) change the resistance thus current and thus improve resolution of the 
top part of the gel (bottom of the autorad if you read downwards. 
        
However, as well as answers to my question many people suggested adding 1/3 
volume of 2M sodium acetate to the bottom buffer (ie. 400ml TBE 1x + 200ml 
2M Sodium acetate) instead of using wedge spacers. This works just the same 
as the wedge spacers (I tried both on the same material) and is a lot 
easier. Wedge gels take a long time to pour (relative to normal skinny 
ones) and of course use a LOT more acrylamide. The acetate acting as an 
anolyte retards the DNA elution from the gel. I was given the reference for 
this technique but don't have it to hand at the mo. The time you add the 
acetate depends on the length of run you are performing for a single run 
add at the beginning but if running for a long time (ie. the smallest band 
size on the gel is 300-400 bases long) you should add the acetate after 
approx. the first half of the total run time to avoid overcompression.




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