I have a insert of 400 bp cloned in vector pbluescript II KS (+) of size 3.0kb at RE sites XbaI and XhoI. But when I try to double digest the plasmid it is not happening. I am sharing picture of result showing the same. Please can anyone provide me the reason and solution for this.
Fig: Lane1: 100 bp plus ladder; lane 2: plasmid double digested; lane 3: plasmid single digested; lane 4: uncut plasmid
I am assuming when you say "it is not happening" means you are unable to see the 400bp insert in the gel? You do not say how much plasmid you digested but more than likely there is not enough insert in the gel to visualize.
Your 400bp insert is ~12% of the overall 3400bp size of the plasmid so if you digested only 100ng of plasmid, you would only have ~12ng of insert to see in the gel which is on the low end of what is possible to see in an agarose gel. If you double digest 500ng of the plasmid you should be able to easily see the insert (~60ng).
Hello, if you are sure of the efficiency of your cutting enzymes, I think that if you use enzymes that have a greater distance than each other in the MCS area, the digestion process will be done better.
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