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Restriction Endonucleases >
Restriction Endonucleases >
BcgI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the BcgI gene from Bacillus coagulans (H. Kong).
Reagents Supplied:
NEBuffer 3 (10X)
S-adenosylmethionine (SAM) (32 mM)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | Not Recommended | | NEBuffer 2: | | Not Recommended | | NEBuffer 3: | | 100% | | NEBuffer 4: | | Not Recommended |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Impaired by overlapping | | dcm methylation: Not sensitive | | CpG methylation: Blocked by some combinations of overlapping |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.50 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 3
Supplemented with 20 μM S-adenosylmethionine (SAM)
Incubate at
37°C.
1X NEBuffer 3:
50 mM Tris-HCl
100 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
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.
Concentration:
2,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- BcgI cleaves DNA substrates twice to excise its recognition site generating a 32 base-pair fragment with 2-base 3“ overhangs.
- 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.
FAQs
- Why isn't BcgI cutting my DNA?
- Does BcgI require SAM for activity?
- Do BcgI generated ends ligate?
- What is the molecular weight of BcgI?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 10 units of BcgI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 15 units of BcgI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC
released < 0.2% of the total radioactivity.
Reagents Sold Separately
NEBuffer 3
S-adenosylmethionine (SAM)
Companion Products
dam-/dcm- Competent E. coli
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,200,336
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