T4 Phage β-glucosyltransferase (T4-BGT)

Description

T4 Phage β-glucosyltransferase specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5-hydroxymethylcytosine (5-hmC) residues in double-stranded DNA, making beta-glucosyl-5-hydroxymethylcytosine (1,2).

Product Source

An E. coli strain that carries the cloned bgt gene from bacteriophage T4

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer™ 4-2010X
Uridine Diphosphate Glucose-2050X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to protect 0.5 µg T4gt-DNA against cleavage by MfeI restriction endonuclease.

Protection Unit Assay Conditions:
0.5 µg T4gt-DNA, 1X NEBuffer 4 and 40 µM UDP-Glucose in a 30 µl reaction. Incubate for 1 hour at 37°C followed by 10 minutes at 65°C. The extent of protection by T4 -BGT is determined by the addition of 20 µl 1X NEBuffer 4 and 10 units of MfeI. Incubation at 37°C for 30 minutes is followed by analysis on agarose gels.

Enzyme Properties
Activity in NEBuffers:
NEBuffer 1 100
NEBuffer 2 50
NEBuffer 3 50
NEBuffer 4 100% 

Survival in a Reaction: A minimum of 0.16 unit for 16 hours is required to protect 0.5 µg T4 gt-DNA against cleavage by MfeI.

Reaction Conditions

1X NEBuffer 4
Incubate at 37°C

1X NEBuffer 4:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
1 mM DTT
pH 7.9 @ 25°C

Storage Temperature

-20°C

Storage Conditions

20 mM KPO4
200 mM NaCl
0.25 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.0 @ 25°C

Heat Inactivation

65°C for 10 min

Molecular Weight

Theoretical: 40666 kDa

References

  1. Josse, J. and Kornberg, A. (1962). J. Biol.Chem.. 237, 1968-1976.
  2. Tomaschewski, J. et al. (1985). Nucleic Acids Res. 13, 7551-7568.
  3. McNicol, L. A. et al. (1973). J. Mol. Biol. 15, 76,, 285-301.
  4. Szwagierczak, A. et al. (2010). Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 38, e181.

FAQs

  1. Will T4 Phage β-glucosyltransferase (T4-BGT) work in CutSmart Buffer?

Protocols

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

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Interactive Tools

Citations

  • McKernan KJ, Spangler J, Zhang L, Tadigotla V, McLaughlin S, Warner J, Zare A, Boles RG (2014). Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment PLoS One. 9(5), e96492. PubMedID: 24788618, DOI: 10.1371/journal.pone.0096492
  • Bhattacharyya, S., Yu, Y., Suzuki, M., Campbell, N., Mazdo, J., Vasanthakumar, A., et al. (2013). Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer Nucleic Acids Research. DOI: doi:10.1093/nar/gkt601

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.