RE digestion Help needed everyone - (Nov/20/2007 )
I cutted the plasmid pUC with Sal I and EcoRI in individual vials and run the plasmid in 0.7% agarose only one band was seen. I thought may be b,cos of one RE site in it it has shown 1 band so I mixed half the prior mixtures of both SalI and Eco RI and again added both the buffer and the enzyme to the reaction mixture as given below
10 microlitre heated at 45 degree to remove the enzyme of prior reaction
1 microl. of 10 X buffer of Sal I
1 microl. of 10 X buffer of Eco RI
0.5 of enzyme SalI
0.5 of enzyme Eco RI
SDW 6 microl
placed in waterbath at 37 degree for 2-3 hours
Again no digestion as the same DNA band was seen when plasmid run in 0.7% agarose.
Next Experiment which I have done :
I have also done RE digestion of Lambda DNA [2 microl.]with Hind III [0.5 microl.] ., 10 X buffer [1 microl.] where I got the result {with clear digested band } but ligation of the same digested DNA (5 microl.) was not seen . As I firstly heated it {digested sample} at 45 degree 5 min and then placed it in Table top cooler and then added 10 X ligase buffer 2 micl. Ligase enzyme 1 mic. place at 16 degree overnight. and the next day added gel loading dye to it and run in 0.7% gel. As usual No result.
Plz help me
10 microlitre heated at 45 degree to remove the enzyme of prior reaction
1 microl. of 10 X buffer of Sal I
1 microl. of 10 X buffer of Eco RI
0.5 of enzyme SalI
0.5 of enzyme Eco RI
SDW 6 microl
placed in waterbath at 37 degree for 2-3 hours
Again no digestion as the same DNA band was seen when plasmid run in 0.7% agarose.
Next Experiment which I have done :
I have also done RE digestion of Lambda DNA [2 microl.]with Hind III [0.5 microl.] ., 10 X buffer [1 microl.] where I got the result {with clear digested band } but ligation of the same digested DNA (5 microl.) was not seen . As I firstly heated it {digested sample} at 45 degree 5 min and then placed it in Table top cooler and then added 10 X ligase buffer 2 micl. Ligase enzyme 1 mic. place at 16 degree overnight. and the next day added gel loading dye to it and run in 0.7% gel. As usual No result.
Plz help me
Find a buffer that is compatible for both Sal I and EcoRI, then set up three reactions: 1. with both enzymes 2. with just Sal1, 3. with just EcoRI. You should see two bands in 1 and one band in each of 2 and 3 if your enzymes are cutting properly.
I'm a bit confused but according to NEB you can use the EcoR1 buffer for a double digest of Sal1 and EcoR1. You should be able to digest in one step but are you purifying the digested vector before attempting ligation?? Even if you heat inactivate the enzymes, you may need to gel/column purify before a ligation will work. Are you quantifying your products on a gel and by OD before the ligation? How much DNA are you putting into the ligation reaction? I've found that less is more (ie better results with only 25-50ng vector). You may not necessarily see two bands in the digested vector. It depends on how much you are cutting out. If both sites are in the mcs and only separated by say 20 bases, you'll never see the 20 bases and only visualize one band. Definitely include digests of each individual enzyme (in EcoR1 buffer). That way if you have a digestion problem you will know which enzyme is to blame.
10 microlitre heated at 45 degree to remove the enzyme of prior reaction
1 microl. of 10 X buffer of Sal I
1 microl. of 10 X buffer of Eco RI
0.5 of enzyme SalI
0.5 of enzyme Eco RI
SDW 6 microl
placed in waterbath at 37 degree for 2-3 hours
Again no digestion as the same DNA band was seen when plasmid run in 0.7% agarose.
Next Experiment which I have done :
I have also done RE digestion of Lambda DNA [2 microl.]with Hind III [0.5 microl.] ., 10 X buffer [1 microl.] where I got the result {with clear digested band } but ligation of the same digested DNA (5 microl.) was not seen . As I firstly heated it {digested sample} at 45 degree 5 min and then placed it in Table top cooler and then added 10 X ligase buffer 2 micl. Ligase enzyme 1 mic. place at 16 degree overnight. and the next day added gel loading dye to it and run in 0.7% gel. As usual No result.
Plz help me
Find a buffer that is compatible for both Sal I and EcoRI, then set up three reactions: 1. with both enzymes 2. with just Sal1, 3. with just EcoRI. You should see two bands in 1 and one band in each of 2 and 3 if your enzymes are cutting properly.
Dear Smu
I have seen the chart given by the company where both the enzyme work at a relative activity of 100 being of Eco RI where as Sal shows an activity of 75 % in the buffer A. Does it have any problem.
Dear rkay
Should I ppt the reaction mixture containing the digest with phenol/ ethanol pptation and then dissolving in TE buffer . carry out electrophoresis to see the conc. as done in PCR. and then take the appropriate digested DNA in ligation reaction mixture my is a 10 X ligation buffer but it is not homogenous it shows particles {?/??///}
sallie
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Dear Smu
I have seen the chart given by the company where both the enzyme work at a relative activity of 100 being of Eco RI where as Sal shows an activity of 75 % in the buffer A. Does it have any problem.
[/quote]
I guess that it really depends on what you're doing. If you don't need 100% double digestion of your plasmid then I would go ahead and put both enzymes together knowing that only 75% of your DNA will be cut with both enzymes. If you need 100% of your plasmid cut, then perhaps the thing to do is cut with one enzyme, then adjust the buffer composition and add the second enzyme. You should be able to find buffer compositions in most catalogs. If this doesn't seem possible, then cut with one enzyme, purify the DNA (Geneclean kits work good for this), and then cut with the second enzyme.
what band sizes do you want to resolve?