What can possibly go wrong in a simple PstI digest?
reaction component :
DNA(50ng/uL) 4 uL
10XNEBuffer3.1 1.2
PstI(20u/uL) 0.4
ddH2O 6.4
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1) DNA is storage in -20oc freezer, and diluted with ddH20 to reaction concentration.
2) 37oc, overnight digestion
3) The same gDNA is well digested with MseI.
if the dna cuts well with other enzymes then it is worth checking the enzyme by cutting another dna ( plasmid or lambda or genomic) with the Pst1 to check the integrity of the enzyme
@Paul Thank you for your advice! I also try to cut pUC19 with PstI , and found it successfully digested. Furthermore, I purified the gDNA and eluted with H2O to ensure there's no inhibit factors.
According to NEB, your enzyme is salt-sensitive so make sure the DNA is 25% or less of the final reaction volume. Since you know the enzyme works, you could try increasing the reaction volume. I think NEB recommends a 50 microliter digest. Also, make sure you vortex the final reaction before the digest to make sure the enzyme is well-mixed into the solution. I've seen digests fail due to lack of mixing.
following on from Katie'answer mixing of the buffer is very important.When frozen buffers thaw the salt melts out first and ice floats on top.Then the rest of the ice melts and forms a water layer on top of the concentrated buffer so there is a risk of taking either water or double concentrated salts if you do not mix the buffer well before pipetting. I attach a generally interesting troubleshooting sheet from NEB for anyone with similar problems to see but I have very little of help to offer. I assume that the od260/280 shows that there is not too much protein still attached to your dna
You can read those few pages online.
http://www.plantsciences.ucdavis.edu/dubcovsky/plb161a/restriction_electrophoresis.pdf
https://www.researchgate.net/publication/264472409_Isolation_and_Characterization_of_Chitinase_from_Tomato_Infected_by_Fusarium_oxysporum_f_sp_lycopersici/figures?lo=1&utm_source=google&utm_medium=organic
... Whether you are student, or a researcher, this might be of some interest to you. Not as an answer to your question, but as a source of ideas to consider.
Good luck.
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