Any experience with Fermentas FastDigest enzymes?

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Any experience with Fermentas FastDigest enzymes?

Postby lcdcracker » Apr 26 2007 4:57 pm

My lab has recently bought several Fermentas FastDigest restriction endonucleases. I have used BamHI and XhoI to successfully cut plasmid DNA with 1-hour incubation (was told not to go for less) and would like to know if PCR products will digest with suitable results. My supervisor doesn't trust these enzymes at all and is strongly discouraging their use on amplified DNA (although she hasn't worked with the enzymes herself), but I'd like to give it a try, since exchanging buffers with our cleanup kits results in low DNA recovery.
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Postby relaxin » Apr 26 2007 5:41 pm

Your supervisor is right, and that is experience. We old-timers only trust what we have experience with. There are two companies whose restriction enzymes are good enough for cloning purpose. The rest is just cheap.
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Re: Any experience with Fermentas FastDigest enzymes?

Postby mchlbrmn » Apr 27 2007 10:50 am

lcdcracker wrote: but I'd like to give it a try, since exchanging buffers with our cleanup kits results in low DNA recovery.

I don't understand what the brand of rest. enzyme has to do with purifying the PCR product?
I thought Fermentas was a reputable company. The biggest RE company in my area resells some of Fermentas's REs that they don't produce themselves.
I have sometimes done a shorcut and successfully digested the PCR product without purifying it from the PCR mix. I don't recommend this. Even if the RE you use is compatable with the conditions of the PCR buffer, prolonged incubation with the PCR polymerase in an exhausted PCR mix, especially if you diluted it, can potentially cause some enzymes to run backwards and have nuclease activity. Commonly available purification kits for PCR products now are very reliable with good yield.

Oh, perhaps the "FastDigest" products are intended to work in the PCR mix? If so, I'd trust them myself if you've had problems purifying. However, this may not be the best diplomatic choice in your current employment situation(?).
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Re: Any experience with Fermentas FastDigest enzymes?

Postby relaxin » Apr 27 2007 2:17 pm

mchlbrmn wrote:I don't understand what the brand of rest. enzyme has to do with purifying the PCR product?


Some companies have better QC on their RE than the others. I have seen enzymes which can chew back 14 bp from the cloning site. Trace amount of nonspecific nucleases in the RE can be a problem in cloning, if long digestion time is used.
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Postby phage434 » Apr 27 2007 9:06 pm

Most NEB enzymes digest much more quickly than most people think (probably the others do as well). In 15 minutes most of the action is over.

Adding RE's directly to a PCR mixture is a very bad idea if you are cloning the fragment. The PCR enzymes are almost certain to still be around, and if there are dNTPs, a fill in reaction will take place. If they are exhausted, then exonuclease activity can occur. You want the PCR products purified before cutting. This makes no difference, of course, if you are doing the cutting for diagnostic purposes rather than cloning.
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