how to do serial digestion with EcoR1 and bamH1 - (Apr/08/2008 )

hi,
i want to digest my fragment with both EcoR1 and BamH1 but because of diferent buffer conditions of NEB cataloge, now i want to do serial digestion with these two enzymes. I want to know if digest my frament with EcoR1,how can i remove the buffer of ecor1 from solution to go head with second enzyme,i can heat inactivate enzymes,what about the buffer.
please suggest me in this issue.
SVRREDDY
Ph.D student,
Italy

You could use a DNA purification kit such as Promega's Wizard kit (PCR clean-up kit).

Are you sure you can't use both enzymes together, I'm sure I've done that in the past. Check the NEB buffer activity chart, I'm pretty sure both enzymes work pretty well in any buffer.

http://www.neb.com/nebecomm/tech_reference...ion_enzymes.asp?

Otherwise you could use QIAgen gel extraction kit, without having to run the gel to remove enzymes, just follow the protocol, or an Amicon column.

hi almost doctor,
ya ur right common buffers for both ecor1 and bamh1 are 2,3,4, but for EcoR1 they sent us separate buffer written as EcoR1 buffer,in catalogue they written we used to send separate buffers if, this buffer is not coinsideing with normal NEB buffers,may be i m thinking because of this i m unable to cut in Double digestion.
this is the problem im facing with two different buffers for different enzymes.
if u have suggestions plz send to me.
SVRREDDY

EcoRI cuts in almost any buffer. The only reason for a special buffer is that there is a slight tendency for star activity in normal buffers (cuts AATT sequences sometimes when it should not). This is infrequent and rarely (never in my experience) a problem. We routinely do double digests (hundreds) in buffer 2. Keep the glycerol content of your digestions low (< 5% for sure) and err on the low DNA concentration side.

Something to watch out for with the EcoRI buffer is the presence of Triton X-100, which is highly toxic during chemical transformtion. If you try to cut DNA in EcoRI buffer and add it directly to a ligation mix, and then transform with the ligation mix, your transformation efficiency will be very very low. The other buffers do not have this issue.