Freiburg07/NgoMIV MEBinfo
From 2007.igem.org
Source:
A E. coli strain that carries the NgoMIV gene from Neisseria gonorrhoeae MS11 (M. So).
Reagents Supplied:
NEBuffer 4
Enzyme Properties
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| NEBuffer 1: | <img src="../images/spacer.gif" width="2" height="1" alt=""> | 100% |
| NEBuffer 2: | <img src="../images/spacer.gif" width="2" height="1" alt=""> | 50% |
| NEBuffer 3: | <img src="../images/spacer.gif" width="2" height="1" alt=""> | 10% |
| NEBuffer 4: | <img src="../images/spacer.gif" width="2" height="1" alt=""> | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
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| dam methylation: Not sensitive |
| dcm methylation: Not sensitive |
| CpG methylation: Blocked |
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.13 unit(s)
Reaction & Storage Conditions
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Reaction Conditions:
1X NEBuffer 4
Incubate at 37°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
One unit is defined as the amount of enzyme required to digest 1 µg of Adenovirus-2 DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
Adenovirus-2 DNA
Storage Conditions:
10 mM Tris-HCl
50 mM NaCl
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
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General notes:
- NgoMIV is an isoschizomer of NaeI.
FAQs
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- <a href="faqproductR0564.asp#973">Is this the same as NgoMI?</a>
- <a href="faqproductR0564.asp#974">Is NgoMIV affected by methylation?</a>
- <a href="faqproductR0564.asp#975">How does NgoMIV differ from its neoschizomer, NaeI?</a>
- <a href="faqproductR0564.asp#1243">Does NgoMIV exhibit reduced activity on supercoiled DNA?</a>
- <a href="faqproductR0564.asp#1244">What is the activity of NgoMIV at 25°C?</a>
Quality Control for Current Lot
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Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 20-fold overdigestion with NgoMIV, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with NgoMIV.
A 50 μl reaction containing 1 μg of DNA and 90 units of NgoMIV 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 100 units of NgoMIV with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of NgoMIV with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 20% conversion to RFII as determined by agarose gel electrophoresis.
An appropriate vector is digested at a unique site within the lacZα gene with a 10-fold excess of enzyme. The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates. Successful expression of β-galactosidase is a function of how intact its gene remains after cloning, an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony. Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
