how to do double digestion with EcoRI and BamHI - (Feb/20/2008 )
Hi,
how to do double digetion with EcoRI and BamHI, for double digestion i am using same enzymes of NEB company but they are not working. if any one used those enzymes in the past, if yes please tell me the process how to do double digestion with these enzymes. they given separate buffers and written dd in buffer chart. i checked my plasmid also i mean it contains GUS and Km ,i did pcr to confirm that my plasmid having those or not. actually i want to separate GUS fragments from this construct which is having sites for BamH1 and EcoR1.can any one help me in this issue.
thanks in advance.
SVRREDDY,
Ph.D student,
Italy
You didn't really say which buffer you have been using but a double digest with EcoRI and BamHI requires the EcoRI buffer. Also, BamHI needs BSA so make sure you are adding this into the digest. Otherwise, I'm not sure where your problem could be. Is your plasmid just linearizing or is there no digestion at all? You'll need to give a little more info in order to really help but you can also try each digestion alone (EcoRI only versus BamHI only). This can tell you which enzyme is giving you trouble. Maybe one of your enzymes is no good. Can you borrow a little from a neighboring lab? A better description of what you expect to see versus what your digest produces will help get more specific advice but hopefully this helps a little.
EcoRI works in all buffers and shouldn't be a problem to digest. BamHI is also a good cutter. So is it possible that the enzymes have gone bad. ?
If you go to the NEB website, you can find their recommended buffers for double digests.
Could you write down your digestion formulation?
EcoRI and BamHI are very robust enzymes. As mentioned they work in partically any buffer. I would throw them into NEB buffer 3.
Might you be using too much enzyme?
EcoRI and BamHI are very robust enzymes. As mentioned they work in partically any buffer. I would throw them into NEB buffer 3.
Might you be using too much enzyme?
Hi,
i am using this formulations
Enzyme EcoR1 0.5 microlitre
Enzyme bamH1 0.5 microlitre
buffer 1 microlitre
water 3.0 microlitre
plasmid dna 5 microlitre
i used buffer 3 for this reaction.
thank you very much for your help.
I think the volumes you are using could lead to higher glycerol in the digestion mixture. Try to digest the same amount in say 50 ul (total volume), and use 1 ul of each enzyme. or use the same amount of enzyme and digest in higher total volume. I always found that digesting even small amounts of DNA in higher volumes is always works.
"they are not working". What do you exactly mean: no digestion whatsoever, only linearisation, extra bands on gel?
If the first or second: try to separately in 2 tubes: see of your enzyme is still fine (can you do a control digest on a standard vector, pUC18 or so to make sure). Or maybe: have you got 100% certainty on the sequence of your plasmid?
if the last: increase your volume as too much glycerol leads to star activity (I know for EcoRI, not sure about BaMHI).
What about the concentration/purity of your DNA? (for purity: A260, A280 and A230:any idea on this?).
NEB also report star activity with BamHI.
Looks like it is too much enzyme/(glycerol which the enzyme some in). Double the volume. Do you know how much DNA you are cutting?
If your DNA is not clean, it won't cut due to contaminants. This problem is handle by dilution.
So recommendation is to increase your digest volume to at least 20ul.
Hi,
I do sequential digestion starting with EcoRI first, then BamHI. EcoRI digestion normally around 30' to 1 hr, then gel isolate. BamHI digestion at more than 2 to 3 hrs, then PCR purify. It's tedious and expensive (using Qiagen gel and PCR purification kit) but it works better than doing double digest, which normally will cause star activity headache for me. Reaction vol is 50 ul and DNA is 1 to 3 ug with 5U/ug DNA of enzyme.
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