Difference between revisions of "Restriction Enzyme Based Cloning"

Revision as of 00:31, 2 May 2009

1. If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB
2. Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.
3. Gel purify both vector and insert (Qiagen kit). Elute in 30 uL
4. Combine 3-6 uL insert with 1-2 uL vector. You want about a 3x excess of insert to vector. Place at 50-65C for 5-10 min to help sticky end binding. Also do a no insert negative control.
5. Add 2 uL ligase buffer (single use aliquots) and water/EB to 10 uL final volume. Add 1 uL T4 DNA Ligase (Invitrogen).
6. Incubate 1h-O/N at 16C (water bath in cold room).
7. Transform 5-10 uL into 50 uL DH5a cells (subcloning efficiency):
   1. Make 50 uL aliquots
   2. Add DNA and mix gently
   3. Incubate on ice for 30min
   4. Heat shock for 20s at 37C
   5. Place on ice for 2min
   6. Add 450 uL LB
   7. Incubate at 37C for 1h with shaking
   8. Plate 500 uL and grow O/N at 37C on appropriate plates
8. Pick several colonies and digest to verify insert. Sequence if necessary.