Restriction digestion - (Sep/21/2006 )
Hello all,
I was trying for some time to double digest my DNA (from a TOPO clone) with SpeI and XhoI . It worked once and I had clear bands. I extracted again DNA (several times) but the digestion is not working anymore. I used different enzyme tubes (to be sure the enzyme is still active); I increased the amount of enzyme used.... etc. I also tried to digest the DNA with other enzymes without any luck. I believe there are some inhibitors in my DNA... but I don't have a clue what to do.
I would really appreciate if someone can help me to solve the puzzle.
Thank you, lorelay
I was trying for some time to double digest my DNA (from a TOPO clone) with SpeI and XhoI . It worked once and I had clear bands. I extracted again DNA (several times) but the digestion is not working anymore. I used different enzyme tubes (to be sure the enzyme is still active); I increased the amount of enzyme used.... etc. I also tried to digest the DNA with other enzymes without any luck. I believe there are some inhibitors in my DNA... but I don't have a clue what to do.
I would really appreciate if someone can help me to solve the puzzle.
Thank you, lorelay
Hi there,
If the DNA is the problem...
There are two things which I would immediately do.
1- try a phenol/chloroforom extraction on the DNA. Do this extraction until the organic/aqeous interphase is clean. Remove supernatant. Percipitate with EtOH and resuspend in TE, or provided you take care of your DNA, in water. EDTA inhibits most manipulating enzymes. Cut some DNA, don't use to much enzyme as that the glycerol coming with the enzyme will inhibit your reaction.
See if the DNA cuts.
if not, gel purify your DNA. Extract the frangment and clean up by column. Make sure your column is dry before eluting. Contamination from the wash buffers will kill down stream enzymatic manipulations.
Best of luck
In addition to perneseblue's suggestions, I would recommend u to digest another DNA prep of a dif. vector and digest using the same enzymes to confirm activity of the enzyme.
IF DNA prep is contaminated, then retransform and grow it and try again.
Thank you for the ideas, I already tried what you suggested and nothing worked. that is why I don’t know what else I could do.
Thank you for the ideas, I already tried what you suggested and nothing worked. that is why I don’t know what else I could do. 
hello
wat i feel abt ur prob is der is somethin wrong wit the purity of ur DNA, may be excessive contaminants dat is preventing the action of the restriction enzymes..have u made sure ur restriction ezymes are active??try digesting them with some other samples to ensure their activity..and also try purifying ur DNA samples either using purification kits or using column if the conventional methods doesnt help..hope u'l get ur results soon
anupama
Sometimes high salt concentrations can inhibit enzyme reactions. I don't know which system you are using to extract your plasmid, but if you precipitate your plasmid then wash it with 70% ethanol it should reduce your salt concentration. Then run the digest as normal. this is probibly a long shot if you are using a mini prep kit, but i have had these problems with pcr products.
Long shot but make sure the restriction enzyme concentration of your digest is <10%. The restriction enzymes are suspended in glycerol which at too high of a concentration can inhibit their action in the digest:
5ul DNA
3ul ddH2O
1ul Buffer (appropiate to enzyme)
1ul Restriction Enzyme