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Frequently Asked Questions about Restriction Enzymes (2)

1. What QC assays Fermentas PureExtreme® restriction enzymes pass?

   See here for Standard Quality Control
    

2. Why Fermentas restriction enzymes are of PureExtreme® quality?

   Only at Fermentas all batches of restriction enzymes should pass a unique test of radiolabeled oligonucleotides (LO) in addition to the common quality tests to ensure the highest quality of enzymes.
    

3. What contaminant activities are detected by the radiolabeled oligonucleotides (LO) test?
   
   The slightest presence of 5'-exonuclease, 3'-exonuclease, non-specific endonuclease or phosphatase contaminants, non detectable by conventional methods are detected in a single LO experiment.
    
4. What is the procedure of the LO test?
   
   The LO tests is "Yes" or "No" test, i.e.:
   The test is performed using 5' ³²P-labeled synthetic 17-mer single and double-stranded oligonucleotides as substrates which don't have recognition sites for the restriction enzyme tested. 4 hour incubation is carried out with respective substrates and 10 units of the restriction enzyme in its reaction buffer.
   Reaction products are separated on a polyacrylamide gel and results are analyzed by autoradiography or storage phosphor imaging. The restriction enzyme tested is considered to be PureExtreme® when no traces of oligonucleotides' degradation are visible.
    
   The LO test using single-stranded and double-stranded synthetic oligonucleotides and restriction enzymes from different vendors.
    
    
5. Do restriction enzyme preparations from major suppliers pass the LO test?

   NO! Though it's a common belief that the quality of restriction enzyme from different vendors is almost the same, not all commercial products are pure against the LO test.
   Labeled Oligonucleotide Test using restriction enzymes from Major Suppliers
   

   Enzyme

   COMPANY

   A

   B

   C

   D

   E

   Fermentas

   ss

   ds

   ss

   ds

   ss

   ds

   ss

   ds

   ss

   ds

   ss

   ds

   BamHI

   -

   x

   -

   -

   -

   -

   -

   x

   -

   -

   -

   -

   BglII

   x

   -

   x

   X

   -

   -

   x

   -

   X

   -

   -

   -

   ClaI/Bsu15I

   X

   X

   -

   -

   -

   -

   -

   -

   X

   X

   -

   -

   EcoRI

   -

   -

   -

   -

   -

   -

   -

   -

   x

   -

   -

   -

   EcoRV/Eco32I

   -

   -

   X

   -

   -

   -

   -

   -

   X

   -

   -

   -

   HindIII

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   KpnI

   -

   -

   X

   -

   -

   -

   X

   X

   X

   -

   -

   -

   NcoI

   -

   -

   -

   -

   -

   -

   X

   -

   -

   X

   -

   -

   NdeI

   X

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   NheI

   X

   X

   X

   X

   -

   -

   -

   -

   -

   -

   -

   -

   PstI

   -

   -

   X

   -

   -

   -

   -

   -

   -

   -

   -

   -

   SacI/SstI

   X

   X

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   SalI

   -

   -

   -

   -

   -

   -

   X

   -

   -

   -

   -

   -

   SmaI

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   XbaI

   X

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   XhoI

   X

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   -

   ss  single-stranded oligo,   ds  double-stranded oligo,   X  contaminated,   x  minor contamination,   -  pure

6. Do cloned enzymes always pass the LO test?

   NO! Though sometimes cloned enzymes may be easier to purify than restriction enzymes of the native origin, the purity is ultimately dependent upon the methods and degree of purification at all cases. Cloned enzymes do not, by definition, pass the LO test.
    

7. Do enzymes pure against Blue/White Cloning assay also pass the LO test?

   NO! Blue/White Cloning assay involves digestion and re-ligation of a vector with a unique restriction enzyme sites within a lacZ alfa gene. Induction of functional beta-galactosidase by IPTG is monitored on X-Gal containing plates by percentage of white vs. blue colonies formed. The passing cut-off point is usually between 2-5% white colonies. Our studies have shown that restriction enzymes with as few as 0.16% white colonies have failed the more sensitive LO test.

Updated lapkričio 29, 2006 13:06