In double digest, the restriction endonuclease enzymes, EcoR1 and BamH1 are used. Does the order of the ezymes used first effect the number of fragmented DNA that is obtained? The DNA used is the lambda DNA.
2006-08-14 16:04:24 · 6 answers · asked by hanson_daren 1 in Science & Mathematics ➔ Biology
OK...Let's say conditions are optimum and both the enzymes are working at it's best. I've checked it out and there are no overlapping of DNA fragments st which both the enzymes cut.
2006-08-17 16:33:31 · update #1
The only way the order might matter is if you didn't change the buffers betwen the reactions (these buffers set up the right conditions for each of the two restriction enzymes to cut the DNA, and they might well not be happy under the same conditions), or if the sites overlapped. Assuming you either find a buffer that's good for both and digest with one, then the other OR with both together, and that they don't overlap, order isn't important. One last caveat: both enzymes must be active and not have their 'star activity' activated. If one is less active, it'll be stimulated by finding shorter fragments. If one (BamH1 is notorious for this) has star activity, if the buffer conditions aren't just right, it will cut where it should not cut and confuse the pattern.
2006-08-19 09:54:53 · answer #1 · answered by Lorelei 2 · 0⤊ 0⤋
The answer depends on why you are asking. If this is a homework question about restriction endonucleases, that would have an answer. If this is a question about actually using these enzymes in the lab to digest lambda DNA, then that would have another answer. In practice, one needs to consider the total amount of DNA that will be cut, the reaction conditions (concentration, double digest vs. sequential digest) and what the purpose of the digest is. EcoRI is notorious for having star activity and can be a pain to use. Different reaction conditions may produce different results.
2006-08-15 02:40:19 · answer #2 · answered by Slackenerny 4 · 0⤊ 0⤋
Unless the two restriction sites are really close together it won't be a problem either way.
In the case of Lamda , the smallest fragment formed would be 1120 nt in length so no problems. Just make sure you use the right buffer or you will get star action occuring. EcoRI cuts in funny places if the right buffer isn't used. You can do a simultaneous double digest with Promega buffer E, or with NEB buffer 2.
2006-08-18 07:55:35 · answer #3 · answered by uselessadvice 4 · 0⤊ 0⤋
you should have a look on the map of the DNA to see if there is any possibility to have different segments in case of using the restriction enzymes in different order. however as i remember i think no... but i am not sure
2006-08-15 02:12:43 · answer #4 · answered by Anonymous · 0⤊ 0⤋
how can you ask without mentioning the parallel 4 helix bundle now you got me all confused ( hey humor ain't wit but i try) and since i'm asking , why are you playing with DNA anyway
2006-08-14 23:41:27 · answer #5 · answered by Anonymous · 0⤊ 0⤋
In theory it shouldn't, but there may be some weird effect in practice.
2006-08-14 23:36:31 · answer #6 · answered by Anonymous · 0⤊ 0⤋