do you sonicate or digest with lysozyme? - which one do you prefer and why.... (Mar/08/2006 )
i have already posted this type of question before, but this time i want to know whether it is better to sonicate or digest with lysozyme (triton etc) the bacterial pellet,....molecular cloning advises against sonication....but some protocols do just sonication....what do you prefer?
we usually do both
thanx aimkins, do you sonicate and then add lysozyme? is that what you mean?
I have tried both techniques, and lysozyme in a freeze/thaw seems to work best. You can also add some DNase I if you're coming from cells.
thanx swanny, yes lysozyme protocol actually says add Rnase DNase.....so ill try both too i think...
Kathy - we take it straight out of Qiaexpressionist (his-tag purification, NiNTA column, preparation of cleared lysates under native conditions)
resuspend (we use about 5 times as much buffer here as you're supposed to; the only major change...seems to make sonication more effective, although it does take longer)
add lysozyme to 1mg/ml, incubate 30' on ice
sonicate (if very viscous add RNAse and DNAse)
spin lysate - go on to column
thanx a lot aimkins, ill do that this time, first time i did only sonication i got big big smear and fat bands i guess of my protein (not very obviuos because of the smear) in the insoluble fraction... ..so i guess i lost my protein here...because in the eluate there were only very faint bands of the protein ....but this smear...you think its DNA? so by adding DNase it is supposed to disappear?
Do you have a French press? I prefer using that to sonication.
um, you saw a big smear on your gel of what? lysate? the final eluate? wash fractions? you haven't, by chance, run a gel of all 3, have you? this can help in troubleshooting...and I would like to know why you think your protein is in the insoluble fraction?
I would not assume a big smear is DNA. we very very rarely use that step.
I used to sonicate, and then to add DNase and RNase because to viscous to be filtrated before affinity column purification.
It s possible that you have DNA