Hi, I am using gateway to get fusion-protein express plasmid.
I use the pLgw EcoDam-V5-RFC1(size is 10.2kb) as the destination vector and do the LR reaction follow the protocol. but after that , I used the restrict enzyme to cut the plasmid and check the size(I have sequenced the insert DNA sequence is correct before) but the size is much smaller than expect( fusion plasmid except size around 10k) ,the real size is around 6k, I haven't meet this problem before and don't know how to do, could anyone give me some idea? thanks a lot!
How are you determining your plasmid sizes? If you are simply running them out on an agarose gel, be aware that plasmids can run "too small" or "too large" due to supercoiling.
Check your digest chart. You might have two products that are nearly the same size and running as one band instead of two.
- Badges
- Science topic