Sequential digestion with NheI and XhoI - Restrictive Digestion (Mar/09/2005 )

Hi,

I did my digestion with these two enzyme sequentially with respective optimal buffers. After the first digestion with NheI using Tango buffer, the linear plasmid seemed to be at the right size and when I proceeded with the second digestion, the insert band I expected was in a much lower intensity than that of plasmid.

The recipe for both digestion is as follows.. I did Ethanol precipitation after the first digestion and the DNA was dissolved in TE.

24microL plasmid
3microL buffer
3mcroL enzyme

Incubation was in air incubator (not water bath) at 37C for 1 hour for each digestion.

I would be very happy if you guys can give me some suggestion what can be the problem.. I am a bit desperate now!

thanks

hi
i suppose you vizualize your digestion products on a Ethidium bromide agarose gel. As the relative intensity of bands is proportionnal to the mass of nucleic acid, iyour result is not suprising.

Let say your plasmid is 5000PB
After two digestion, your insert is 500pb.
It means that :
for 1 molecule of plasmid digested you get 1molecule of insert
fo 1gram of plasmid digested you'll get 1 x 500/5000 gram of insert. That is 10fold less.

So on a gel after the digestion the band of the insert is obviously less intense.

Moreover, assuming that not all the plasmid is digested, i's possible that twe linear and the linear digested form of the plasmid are on the same band. That is an oher desequilibrium betwen the two relative intensities..

I hope i m understandable...


Moreover you wrote that you used 3µL of enzyme. I'm not sure, but it seems quite a lot for a digestion. Personnally, i use 0.5 µl of NEB-supplied enzyme for this. But it depends of the concentration of your plasmid prep. But if you really need this quantity of enzyme, maybe your reaction volume is too little.

Finally, maybe you get too much glycerol by adding an enzyme volume 10% of total reaction volume...


Cheers.
Fred

hi
the total reaction time depends on the efficiency of the enzyme. But i would sya that for an efficient reaction 2hours are the minimum.

Briefly NEB provise helpful guidelines to set up a restriction assay. Here is the link :
http://www.neb.com/nebecomm/tech_reference...up_reaction.asp


and restriction endonuclease overview :
http://www.neb.com/nebecomm/tech_reference...es/overview.asp

I forgot to tell you yestrday : but after a first digestion and precipitation, i resuspend in ddH2O instead of TE, due to the fact TE contains salts and all salts needed by the enzyme is provided normally by the buffer. i don't know the tango buffer but i think it's should be the same protocol as for the neb ones i use. Maybe you get too much salts in your reaction media and this
1 can disturb restriction enzyme and
2 too much salts disturb the migration on agarose gel (little smearing and wrong size


Finally, for the quantity of enzyme, i usually take 0.5µl of enzyme for volume up to 30µl and quantity p to 5µg of plasmid and let 2hours
For more plasmid and or more reaction volume i take 1µl of enzyme
Neb enzymes are at 10 000u/ml 50%glycerol. Normally, one unit is defined as the amount of enzyme required to digest 1µg of lambda DNA in 1h at 37°C in a total voume of 50µl. Hence, by taking this quantities, you are ok.



Here i explain what i wrote yesterday :
after your first digestion, you get the linearized 4500 bp plasmid.
After the second one you get the "linear digested" 4000bp plasmid (assuming that as wrote yesterday your fragment is 500bp)
There is no chance that your plasmid forms a concatemerized molecule (no ligase for the first reason)

Hope you'll succeed soon

Fred