I am a starter for cloning. I need help. Thanks
Purpose: Insert the plasmid (1230bp) into vector (6650) by Ecor1 and Xho1 REs
Procedure: 1. PCR (primer design with ECOR1 and Xho1 at the end and another 6 bp for protecting the RE site)
2. Run the DNA gel, see clear single bands, and gel recovery under UV light
3. Digest the PCR product and vector with ECOR1 and Xho1 together since the same buffer system for both REs. 4h 37 oC. 65C deactivate. Then add CIP for both vector and PCR product 37C 30 min.
4. Run DNA gel, see clear bands, then do gel recover under UV light.
5. Ligation: T4 ligase (invitrogen) 16 C overnight, ratio between insert and vector was 3:1, 6:1, 9:1.
6.Transformation: DH5alph competent cells, heat shock 42 C, 90 sec. agar plate with carbenicillin. (resistance amp)
Results: 1) set up positive group, transformation the vector without REs (circular vector), get colonies
2) set up a gourp, which only cut the vector with one RES either ECOR1 or Xho1, then ligation. After transformation, do not get any colonies
3) others ligation product do not get any colonies.
Problem: 1. UV harm the DNA?
2. 5x ligation buffer (invitrogen) has PEG, is it ok for ligation system?
3. Do I need do phenol-chloroform participation instead of gel recovery?
How fix it?