BcgI

Description

Product Source

An E. coli strain that carries the BcgI gene from Bacillus coagulans (H. Kong).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
S-adenosylmethionine (SAM)-2032 mM
NEBuffer 3.1-2010X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

Reaction Conditions

1X NEBuffer 3.1
Supplement with 20 μM S-adenosylmethionine (SAM)
Incubate at 37°C

1X NEBuffer 3.1:
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
100 μg/ml BSA
pH 7.9 @ 25°C

Activity in NEBuffers

NEBuffer 1.1: 10%
NEBuffer 2.1: 75%
NEBuffer 3.1: 100%
CutSmart® Buffer: 50%

Diluent Compatibility

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 min

Methylation Sensitivity

dam methylation: Impaired by Overlapping
dcm methylation: Not Sensitive
CpG Methylation: Blocked by Some Combinations of Overlapping

Notes

  1. BcgI cleaves DNA substrates twice to excise its recognition site generating a 32 base-pair fragment with 2-base 3´ overhangs.
  2. Requires S-adenosylmethionine for optimal activity (supplied with enzyme). S-adenosylmethionine is stored at -20°C as a 32 mM solution dissolved in sulfuric acid (0.005 M) and 10% ethanol. SAM in this solution stored under ideal conditions remains active for up to 6 months. SAM is unstable at pH 7.5 and 37°C and should be replenished for reactions incubated longer than 4 hours. Many problems in achieving complete digestion can be alleviated by using fresh SAM.
  3. May exhibit star activity in NEBuffer 2.1, or CutSmart Buffer.

FAQs

  1. Why isn't BcgI cutting my DNA?
  2. Does BcgI require SAM for activity?
  3. Do BcgI generated ends ligate?
  4. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  5. How can I access the old NEBuffer Activity Chart?
  6. Why is my Restriction Enzyme not cutting DNA?
  7. Why do I see additional DNA bands on my gel after a restriction digest?
  8. Why do I see a DNA smear on an agarose gel after a restriction digest?
  9. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

Protocols

  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes

Selection Charts

Feature Articles

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

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.
  • 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.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

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