Help with AVRII digestion - (Oct/25/2006 )
I have a plasmid I need to cut with AVRII which should result in a ~7.5 kb fragment and an ~ 900 bp fragment. After running out the digestion on a .75% gel, I get strong bands at ~ 8kb and very faint bands at ~1000 bp suggesting the AVRII is not doing such a great job at cutting the plasmid. How many units of AVRII should I be using in a 30 uL reaction with ~ 1.5 ug of DNA? I've read some people have to use an excess of AVRII, but never really give an amount that works for them. Any suggestions?
Greg
-UnknownG-
QUOTE (UnknownG @ Oct 25 2006, 04:14 PM)
I have a plasmid I need to cut with AVRII which should result in a ~7.5 kb fragment and an ~ 900 bp fragment. After running out the digestion on a .75% gel, I get strong bands at ~ 8kb and very faint bands at ~1000 bp suggesting the AVRII is not doing such a great job at cutting the plasmid. How many units of AVRII should I be using in a 30 uL reaction with ~ 1.5 ug of DNA? I've read some people have to use an excess of AVRII, but never really give an amount that works for them. Any suggestions?
Greg
Greg
If you only see the 8000 and the 1000bp bands, it means that the enzyme cut the whole amount of plasmid you used. Keep in mind that the 1000bp bands represents around 11% of the total amount of DNA (1000/9000), therefore if you cut 1ug of DNA, the 1000bp band will be 110ng more or less, while the 8000bp band will be a bit less than 900ng
-dnafactory-