Ligation and Transformation
- Plan to do a ligation with the vector only and one with vector + insert.
- Dilute vector to 20 ng/ul for use in the ligation, use Millipore pure dH2O.
- 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
- Combine vector, insert, and pure dH2O in a total volume of 8 ul in a 1.5 ml tube
- Add 1 ul 10X Ligation buffer and mix with pipetting.
- Add 0.5 ul Quick T4 DNA Ligaseand mix with pipetting.
- Add 0.5 ul of the appropriate restriction enzyme. Your total volume in the tube should now be 10 uL.
- Let stand 5 min at room temperature.
- Thaw competent cells on ice (eg. 100 ul of Z-competent JM109).
- Heat ligation at 65 C for 10 minutes.
- Place ligation tubes on ice for 2 minutes.
- Dilute competent cells using dilution buffer. Add 70 uL of buffer if you are doing two plates.
- Add half of your competent cell/buffer mix, 60 uL in the case of two plates, to your ligation.
- Let stand on ice for 5 minutes.
- Spread onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly.
- Place your plates into the incubator.