making your own size markers
"Jerry Marshall ", Y
jerry at PICAN.PI.CSIRO.AU
Wed Feb 8 18:25:25 EST 1995
Here's a method for making your own low MW DNA size markers:
Buy, borrow or otherwise procure a microlitre of Pharmacia's 100 bp
ladder. This product is made by partial digestion of a plasmid that
contains lots of 100 bp fragments separated by AvaI sites. Use your
microlitre of the ladder to transform your favourite E. coli strain
(there's still some intact plasmid there since the ladder is only
partially digested). Purify the plasmid and do a partial digest with
AvaI. It helps to do a pilot run taking timepoints to allow you to
optimise conditions for production of a nice ladder (overdigestion gives
a nice plasmid band and a nice band at 100 bp...).
The Pharmacia product is spiked with a DNA fragment of 800 bp to allow
you to orient yourself after you've run your DNA off the bottom of the gel.
This can easily be reproduced by adding a restriction fragment from some
other clone or a PCR product.
The whole procedure is very straightforward and considerably cheaper than
buying the product from Pharmacia.
Thanks to Tony Ashton for the idea.
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