Exonuclease and Phosphotase contamination - (Jul/14/2006 )
Hello,
I was working on protein purification on a restriction enzyme and came to the problem of contamination with exonuclease and phosphotase, which might cause failure in ligation. Can I know what is the main reason that cause this kind of contamination and how can I prevent it during the purifcation?
Thank you very much.
Alys
exonuclease may come from same reasons as DNase RNase... So wear gloves and appropriate sterilized clean material.
I've never heared about phosphatase contamination...
I was working on protein purification on a restriction enzyme and came to the problem of contamination with exonuclease and phosphotase, which might cause failure in ligation. Can I know what is the main reason that cause this kind of contamination and how can I prevent it during the purifcation?
Thank you very much.
Alys
If you meant the phosphatase (CIP) which have to be removed for ligation after the dephosphorylation of the DNA, just use a kit. I always use Qiaquick PCR Purification Kit and never had problems to get rid of the enzymes (which is surely valid for all other kits, too). If you use other phosphatase (e.g. SAP) you can heat inactivate, there's a long thread about this here in bioforum.
hobglobin
New England Biolabs website reports that restriction enzymes are checked for the following contaminants. Obviously, the fact that checking is required means that even the commercial vendors have trouble with this. Exonuclease contaminants are the most troublesome as adding too much enzyme or cutting for more than the minimal amount of time necessary can result in nibbled ends that no longer ligate. A technical rep at another company once admited to me that CIAP and BAP were the most difficult enzymes to eliminate such contaminants from. The contaminants have more trouble working at the higher temperatures that the phosphatases can tolerate, so these modifying enzymes are always used at high temps.
DNA/RNA Modifying Enzymes Quality Controls
Assay for Nonspecific Endonucleases
To assay for nonspecific endonuclease contamina-tion, modifying enzymes are incubated with a supercoiled plasmid substrate. A single nonspecific nick in the RF I DNA converts it to the RF II form (nicked circle). Aliquots are incubated with 1 µg of RF I (supercoiled form) DNA in a reaction volume of 50 µl using the recommended reaction buffer. Incubations are performed for 4 hours at the recommended temperature. The two forms are easily distinguished on agarose gels and the percent conversion from RF I to RF II is reported on the Technical Data Card provided with each enzyme.
Overnight Assay for Nuclease Contamination
All modifying enzymes are incubated overnight in their recommended reaction buffer with 1 µg of substrate DNA (HindIII fragments of Lambda DNA or HaeIII fragments of fx174 DNA) in a volume of 50 µl. The characteristic banding pattern of the DNA substrate is compared to the pattern produced from an excess of enzyme incubated overnight. A sharp, unaltered pattern under these conditions is an indication that the enzyme preparation is free of detectable levels of nonspecific DNases. The maximum number of units that can be incubated overnight is reported on the Technical Data Card provided with each enzyme. Enzymes used for RNA work are tested for ribonuclease contamination by incubation with 5 µg of MS2 RNA in the supplied reaction buffer for 1 hour at 37°C, followed by denaturing agarose gel electrophoresis.
Assay for Exonuclease Contamination
All modifying enzymes are incubated with 1 µg of a mixture of single and double-stranded, 3H-labeled E. coli DNA (200,000 cpm/µg) in a reaction volume of 50 µl. The supplied reaction buffer is used for this test. Incubations are performed for 4 hours at the recommended temperature. Exonuclease contamina-tion is indicated by the percent of the total labeled DNA in the reaction that has been rendered TCA-soluble. The limit of detectability of this assay is approximately 0.05%. The results of this assay are given on the Technical Data Card provided with each enzyme.
Assay for Phosphatase Contamination
Phosphatase contamination is revealed by the loss of radioactivity from a 5´ 32P-labeled eight base deoxyribonucleotide d(T)8, as determined by denaturing polyacrylamide gel electrophoresis and autoradiography. Preparations of enzyme are incubated for 4 hours under standard assay conditions in 50 µl reactions containing this substrate.
I am purifying the restriction enzyme from certain strain of bacteria and thinking that the exonucleases and phosphotases are from the bacteria instead of contamination from the environment.
I have gone through few column chromatography for the purification, where it should be able to remove other nucleases and phosphotases. Instead, I still get those contaminants.....