DNA Ligation Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Performing DNA Ligations for Molecular Cloning - Ligation Protocol

1. run the vector and insert on an agarose gel along with DNA ladder
2. quantitate the amount of vector and insert present:
for 6uL of 1/16x diluted DNA ladder, 375ng of DNA is present on the 1650 band (Gibco)
estimate the amount of the vector and insert DNA relative to the 1650 band
also take into account that DNA twice as long as the 1650 band should be twice as bright if the DNA that is double in size is twice the amount of the 1650 band
3. use 200ng of the vector; if the vector is 10kb and the insert if 1kb, add 1/10th the amount of insert to the ligation reaction
4. in one ligation tube, add 3x the concentration of the insert as normal
5. include a control ligation tube with no insert

insert x uL
vector y uL for 200ng of the DNA
T4 DNA ligase buffer 1 uL
T4 DNA ligase 0.5uL
water up to 10uL

larger volume of ligations may be used if the vector or the insert is not concentrated enough

incubate either 14 or 16 degrees overnight; room temperature (22 degrees) two hours; 18 degrees 5 hours; if doing room temperature ligations, make sure that the temperature of the room is not too cold or too hot, depending on the time of the year

6. if desired, heat inactivate the enzyme at 65 degrees for 5-10 minutes