double restriction enzyme digestion - not work? (Sep/21/2006 )

hi, everyone

recently, I digest my DNA with double restriction enzyme (EcoR I and Xba I), but unfortunately, i can not get the target band. They don't work with my plasmid DNA.

I transformed and extracted the plasmid DNA again, however, it doesn't work either.

I don't know why. Maybe these two enzyme are both affected by the methylation of the plasmid DNA?

Please give me some suggestions. Thank you very much!

hi

a few suggestions dont know how relevant

if ur doing double digestion , do u use a single step with one common buffer?
if so did u check if it is compatible with both enzymes ?
are these enzymes bought from the same company?

what is the temperature used? does both have the same temperature requirment?


if u r using double digestion in two steps , how do u add the second enzyme?
after purifying or directly adding the next enzyme?

maybe during purification u lost the concentration?


how is the template DNA concentration?
when working with plasmids of low copy number we will need to use larger amount of PLasmid DNA to see the band clearly , i had that problem with a binary vector in agrobacterium.

give clearer picture about what u did maybe
then we can suggest more solutions

hope it helps
regards
laxmi

Do both of the enzymes linearize the plasmid when used by themselves?

Personal sugestion if digesting a pcr product do an ethanol wash before digesting.
(precipitate overnight then wash with 75%ethanol.)
Also if a 2 step digestion is performed wash the sample inbetween digestions otherwise salt concentrations become too high.

If you are using NEB enzymes.
Use NEB buffer 2 and BSA

Digest should be (100ul)

10ul NEB buffer2 (10x)
1ul BSAS(100X)
1ul EcoRI enzyme
1ul XbaI Enzyme
87ul sample and dH2O

incubate at 37 degrees for a minimum of 2 hrs

Thank you for your advice!

in my experiment, only one step was adopted and the two enzymes were both purchased from MBI company and the buffer was compatible with these two enzymes.

XbaI is sensitive to methilation but EcoRI is not. I would suggest to try with one enzyme first (probably EcoRI), have a look at the gel and, if it works, to use XbaI. If only the second doesn't work, try to transform dam- bacteria with your DNA

Thank you very much! I will try it soon.