Home Protocols Protocol for Glucosylation and digestion of Genomic DNA using AbaSI (#R0665)

Protocol for Glucosylation and digestion of Genomic DNA using AbaSI (#R0665)

For efficient digestion of genomic DNA containing 5-hydroxymethylcytosine, it is necessary to glucosylate (with T4 Phage β-glucosyltransferase (T4-BGT, NEB #M0357) before the cleavage reaction with AbaSI.

Step I: Glucosylation of 5-hydroxymethylcytosine

For a standard reaction use the following conditions:
gDNA 1 μg
10X CutSmart Buffer 2 μl
UDP- Glucose 2 mM 0.4 μl
T4-BGT (10 U/ μl) 1 μl
Sterile water variable
Total 20 μl

Incubate at 37°C for overnight. Purify treated DNA using standard phenol chloroform extraction followed by ethanol precipitation method and resuspend the DNA in an appropriate volume of sterile water for next step. Measure DNA concentration.

Step II: AbaSI digestion

Uses purified genomic DNA from Step I and follow the conditions below:

DNA ~1 μg
10X CutSmart Buffer 2 μl
AbaSI (10 U/μl) 2 μl
 DTT 1 μl
Sterile water variable
Total 20 μl

Incubate at room temperature for 1 hour. Purify DNA by phenol chloroform extraction followed by ethanol precipitation method.

Purified genomic DNA is ready for downstream protocols.