The digest constitution are as follows:
water - 6.5 microlitre
buffer- 1 microlitre
enzyme - 0.5 microlitre
PCR product - 2 microlitre
You do not say how much DNA is in your pcr band but I would run a small volume 1ul on a gel and if the enzyme would cut it into 2 fragments then use enough so that each fragment is visible. You muct realise that short fragments of DNA do not trap much etbr so the same amount of DNA gets weaker if it is cut into shorter fragments. In your example if 2 ul gives a strong band and the enzyme cuts it into 2 fragments then try cutting 4ul to get strong bands and if one of your bands is less than about40 bases then it may still be very faint.Can we see a picture please?
Do you suggest I increase the volume of the pcr product and reduce the water . I would have loved to show a picture, but it was too faint and the resolution of my device could not pick it well.
Yes just cut more product. also do not worry about smearing. Cut pcr product does not smear and partial digests just leave uncut whole amplifier. this is not genomic DNA where cutting is random and all sizes are produced. If your product cuts in one position and is linear then you can only get the 2cut fragments and maybe some whole pcr product in the case of partial digestion or pcr product which is polymorphic at the cut site. Also always run a small uncut sample to make sure that you are separating cut from uncut in your gel as well as the size standard
Dear Ubong Akpan,
As Paul Rutland said, you should slightly modify you digestion protocol. In our lab. we use the following protocol for each 20microliters reaction:
PCR product - 10 microlitre
Enzyme_ 1microliters
buffer -5 microliters
Nuclease Free water-4 microliters.
in my experience this protocol works efficiently
good luck
Hi there!
I would just like to add a few points apart from what already suggested. If you go through NEB website, you'll find that there is a correlation between the amount of DNA intended to digest and the volume of the reaction mixture. 50uL is required per microgram of DNA to be digested ( I too learnt it from researchgate, follow the link given!). Less water in the reaction mix will unnecessarily cause too much molecular crowding to hinder proper reaction. To ensure proper and complete digestion, I think you should have an estimate of the concentration of your PCR product. Accordingly you can use as much Units of enzyme as per your requirement. Then you can load small volume of the digested mix to check for your bands. The rest of it is available for you to proceed with your desired downstream process. As Paul suggested, do run a molecular weight marker and an uncut PCR product to compare.
Hope it helps!
https://www.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions
https://www.researchgate.net/post/How_to_decide_the_volume_of_restriction_digestion_reaction
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