problem in double digestion - (Feb/06/2009 )
Hi friends,
I have isolated a plasmid ,run the gel, observed band at approximately 10 kb.but actually my vector size is 3.5kb,insert size 1kb. that means, I should expect the band at 4.5kb.right, I tried to double digest the plasmid, but could not. what do U suggest.should I go for transformation,plasmid isolation until I get 4.5 kb band or go ahead.
any suggestions are appreciated.
Thannx a million tons
novagen on Feb 6 2009, 08:21 AM said:
I have isolated a plasmid ,run the gel, observed band at approximately 10 kb.but actually my vector size is 3.5kb,insert size 1kb. that means, I should expect the band at 4.5kb.right, I tried to double digest the plasmid, but could not. what do U suggest.should I go for transformation,plasmid isolation until I get 4.5 kb band or go ahead.
any suggestions are appreciated.
Thannx a million tons
Don't go ahead till you get a ~4.5 kb band on gel. There is no point in spending time in downstream steps till you solve this.
The supercolied plasmid band (that constitutes the most of plasmid DNA in a fresh plasmid prep) may be smaller than marker band linear DNA, but not larger.
Did you cut out anything from the original vector. If not, then you could run the uncut original vector and the DNA from the ligation. There should b a difference in the sizes if it has your insert. You could also try to digest using anhy combination of enzymes to get you the confirmation. But donot go ahead with it, till you confirm the presence of your insert.