Does anyone know how long should be sufficient for digestion of a 4kb pcr fragment? in the manual mentioned 1-16 hours, but I don't have sufficient product to optimize all possible hours.
As every enzyme reaction it strongly depends on the amount of substrate, means DNA in this case, you use. In addition, as Ravi Babu mentioned, it matters if the enzyme has star activity which is normally explained at the supplier website. Also it matters whether you a a double or single digest. To my experience, if you use less than 4µg of DNA a digest of 4h is sufficient for single and double digestion.
Hi! Farhat,
It all depends on which enzymes you would like to digest with. Enzymes like ECoRI need max. 2hrs, more than that leads to star activity. So,check the time needed based on the enzyme you choose and with most of these RE, 1 unit of it can cut 1ug of DNA in 2-3 hrs at optimum temp in most cases 37degreeC.
Good Luck,
Madhavi
Some companies now offer Fast Digestion enzymes that reduce the time of the digestion really significant, so check also this option this option!
Good luck
Spiros
Hi Farhat,
Depends on the number of restriction sites presents in your sample.
For optimal results I would advise you to use Fast Digestion enzymes (as suggested by Spiros) with time between 4h to O.N..
Good Luck!
if you dont have sufficient amount of DNA to optimize, i suggest overnight digestion with addition of BSA IF the purpose is "diagnostic" (i.e. to check if this is the right sequence). If the purpose is to clone, i highly recommend 3-5 hours (4 h is a good guess), also with BSA (check Maniatis or New England Biolabs for final concentration, usually supplied at 100x conc.). When digesting for cloning, it also depends how many nucleotides separate the restriction site (which is added by pcr primers) from the end of fragment. There is a chart from New England Biolabs, showing which enzymes require 2 or 3 or 6+ bases from the end of pcr fragment for digestion to work well. Also if the sequence is AT or GC-rich. Sticky ends tend to degrade if the restriction digests are incubated too long. Sometimes you also need to heat-inactivate the enzyme, as it likes to hang on the DNA end, preventing ligation. Remember that if one of the 2 pcr ends does not need to be digested (blunt end), it may be helpful to use 5'-phosphorylated primer to improve ligation efficiency. Never add more than 10% of restriction enzyme to restriction digest reaction, as 10% glycerol in the enzyme stock will start inhibiting the digest (and you will not see this as you're digesting the sites at the end). Collectively, 4h at right temp is a good guess, not more than 1 microliter of enzyme in 20 microliter restriction rxn is good guess, use digested fragment for cloning right away, as sticky ends degrade if stored at 4C, frozen-thawed or overincubated. If your pcr fragment was generated with 3'->5' proofreading enzyme (like Pfu or Pwo) it is a good idea to read instructions well, clean up pcr rxn with qiagen or similar kit (we used to use phenol 20+ yrs ago), as carry-over of enzyme into restriction digest may destroy the ends of your pcr fragment due to residual chewing-back activity in the absence of dNTPs. I would use Fast digestion system only if you're familiar with it, being more conservative helps to achieve more predictable results, many good innovations require reading small print in instructions, some are counterintuitive :) Good luck
Depending on the activity of the Enzyme, I usually take one hour only to cut a 2kp.you have to check the star activity of you Enzyme,
Check out this paper
Article The pJan25 vector series: An enhancement of the Gateway-comp...
Dear Farhat
I am attaching a 4 page chapter about (Restriction Endonuclease Digestion of DNA). Please read it carefully as many factors are involved including time. This protocol says 1 hour incubation. Read the important notes in this chapter and see which one fits your experiment.
Best Regards
What quantity of PCR you have? because it do not depend of the size od the fragment but of the quantity... and the enzyme...
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