I am trying to clone a small 111 bp fragment (using EcoR1 and BamH1 site) into a vector of 7.3 Kb into the same sites.
The protocol I use is:
1. digest the vector sequentially with EcoR1 first, purify by PCR purification kit of promega and then digest using BamH1. Purify by gel extraction.
2. Double digest the vector containing insert
using the same two enzymes using promega buffer E (compatible buffer for double digestion with bamh1 and ecor1). Purify by gel extraction and elute it in a volume of 50 microlitres and then concentrate it by speedy vac to approximatly 20 microl. For the digestion I used 2.5 microgram of DNA.
3. In both the cases the digestion is for 2 hours which I hope is sufficient for complete digestion with 20 units of enzyme per microgram DNA.
4. Since I could not measure the conc of insert bcz of small size I used approximately 1,2, 4 microlitres of insert preparaion for ligation reaction using 100 ng of vector in separate reactions. I used Neb ligase.
5. My vector seemed okay on gel as there is clearly a single nice band. My restriction enzymes are also working as I set up controls with single enzyme.
I couldn't think of other controls to check where the problem lies. Does any one here could help me by providing more tips?
I would like to add upon some more things which I did recently with my vector.
I did a control ligation reaction with my vector cut with HindIII and purified it. Later I used this digested vector for a ligation reaction of 20 microlitres. The electrophoresis showed me a band lower than the lower most band of my original digest without ligase. Simultaneously, there is also a band shift upwards. What is happening here? Pls someone answer the question....