double digestion of plasmid - problem with incomplete digestion (Feb/15/2007 )

Hello,
I have a problem with incomplete double digestion. For 2 days I am trying to digest one nasty plasmid. I checked the single digestions-they work. Check the buffer-compatible with both enzymes. I use 20U enz. per 10ug of plasmid size ~5kb. I added more enzyme and gave it longer time and I still receive this 2 bands close to each other. I mean they are in the right position where the linear band should be, but still there are 2 bands very close to each other.
If someone knows what to do please help me with this nasty plasmid cos it is nasty! Thanks

an

just to confirm - when you do your double digests your total proportion of enzyme in the reaction is still 10% or lower right?
easy to add too much when you have 2 enzymes .....i.e. 5ul enzyme total in a 50ul reaction

10 ug for me sounds too much!
usually u digets 100-600 ngg of plasmid.... more than that can even inhibit the digestion reaction.
try less

Hello,
I have a problem with incomplete double digestion. For 2 days I am trying to digest one nasty plasmid. I checked the single digestions-they work. Check the buffer-compatible with both enzymes. I use 20U enz. per 10ug of plasmid size ~5kb. I added more enzyme and gave it longer time and I still receive this 2 bands close to each other. I mean they are in the right position where the linear band should be, but still there are 2 bands very close to each other.
If someone knows what to do please help me with this nasty plasmid cos it is nasty! Thanks

an

well, it is 100ul reaction, with 20 unit of each enzyme, 10 ul ( 10x buffer) and 10 ug plasmid. as I add and add more enzymes and incubate one of the band seems to gradually disappear, but still I have never had so much problems with any other plasmid than this nasty bastard

well i used to digest 10ug DNA with 20 Unit of enzyme with 10X buffer for one hour, and i have no problem in digestion.
just check out it is not for any methylation problem,
once it happened to me
good luck

I routinely combine 25 µg of pUC vector (14.4 pmol and 8.5 e 12 copies of each unique restriction site) with 20 µL of 10x KGB, 80 units @ of BamH I and Hind III, 3 units of Shrimp Alkaline Phosphatase and Type I water to a final volume of 200 µL. Incubate at 37°C for 3.25 hours. Small scale reactions using single restriction enzymes should be performed and analyzed with uncut plasmid DNA and 5 µL of the vector digest on an analytical 0.8% agarose gel to confirm that both restriction enzymes are performing as expected.

Actual results may vary depending upon the restriction enzymes employed, the number of basepairs between the two restriction sites, and the purity of your DNA.

20 units of enzyme is way too much. Plus 10ug of plasmid is too much as well. Try to reduce it to 2 to 3 ug.

usually i digest 20ug of DNA when i need to seperate some insert from it,
in etoh ppt and recovery from gel i loose a great amount of dna so i start from 20 ug
dna and for confirm digestion in 1 hour i use 40 unit enzyme

Do you have a vector that cuts bands of a know size when digesting with the enzymes that you are using? You could set up an identical digestion using that vector and see if it cuts properly.