DNA Ligation Protocol

DNA Ligation Protocol

1. The vector and insert run on an agarose gel along with a DNA ladder
2. quantitate the amount of vector and insert are present:
for 6UL of 1 / 16x diluted DNA ladder, 375ng of DNA present in 1650 the band (Gibco)
estimate the number of vector and insert DNA relative to the 1650 Band
Also take into account that the DNA twice during 1650 bands to two times brighter when the DNA double in size is double the number in 1650 Band
3. Use of 200ng vector; if the vector is 10kb and insert if 1KB, add 1 / 10th the number of inserts for ligation reaction
4. in one tube ligation, add 3x concentration as usual insert
5. includes control tube ligation without insert.

DNA Ligation Protocol

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

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

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

6. If desired, heat deactivate enzymes at 65 degrees for 5-10 minutes