Ligation and Transformation

Ligation and Transformation

1. Plan to do a ligation with the vector only and one with vector + insert
2. Dilute vector to 10 ng/ul for use in the ligation, use Millipore pure dH2O
3. Calculate the amount of insert needed with the following formula

  10 ng vector x (insert size/vector size) x 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng

1. Combine vector, insert, and pure dH2O in a total volume of 5 ul in a 1.5 ml tube
2. Heat at 42 C for 2 minutes
3. Add 5 ul 2X Quick Ligation buffer and mix with pipetting
4. Add 0.5 ul Quick T4 DNA Ligase(and mix with pipetting
5. Let stand 5 min at room temperature
6. Thaw competent cells on ice (eg. 100 ul of Z-competent JM109)
7. Heat ligation at 65 C for 10 minutes
8. Place ligation tubes on ice for 2 minutes
9. Add at least 20 ul of competent cells to ligation using a cold pipette tip
10. Let stand on ice for 5 minutes
11. Spread onto LB agar plates with the appropriate antibiotic