NEB HF enzymes – regarding library preps

Hi guys,

Just a note about PstI-HF:

The PstI-HF sold from NEB is less processive than the old NEB PstI/red stripe version (go here to see it: .  When digesting ultrapure, high concentration homogeneous DNA (plasmids) digestions fail to go to completion even when left overnight (> 16 hours).  What does this mean for you?  It means that your genomes which are more difficult substrates to deal with will not digest fully unless left to go overnight.  If you want to ensure a good digest use a positive control and switch to the linked product.





4 thoughts on “NEB HF enzymes – regarding library preps

  1. Is the idea for HF that it won’t cut at any other, non-target sites? What makes an enzyme “high fidelity”?

  2. The idea behind NEB’s high fidelity is that your enzyme will have less ‘star’ activity or non-specific nucleic acid cleavage AND you can use one buffer ‘CS’ or buffer 4 + BSA for your digestions, handy if you do double digests. This is rarely a problem since most enzymes are cloned and purification technology is extremely good. Also, you’ll have star activity if you’re dumb about your digestion – i.e. use 100 U of enzyme X to digest 50 ng of DNA.

  3. Interesting! I feel like star activity, even if it occurs, is unlikely to cause much of a downstream problem for GBS library prep, as those fragments that are inaccurately cut are less likely to have adaptors/barcodes ligate to their ends… unless I’m not thinking about this correctly? I’ve ordered some non-HF PstI.

    Another question–how much to do you think the DNA elution buffer could interfere with enzymatic activity? Have you looked at differences in digestion in Qiagen’s “buffer AE”, which contains EDTA, versus just water or Tris?


  4. You’re right about star activity – It’s a problem that won’t disrupt the downstream applications. DNA elution buffer won’t inhibit the enzymes very much. I think NEB recommends large quantities of EDTA to stop restriction digests. TE should have 1mM EDTA and NEB’s stop digestion buffer has 50 mM EDTA.

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