Too stupid to do a MidiPrep? - -how come it is not working- (Oct/30/2006 )

Hey all,

lately I was sort of having a weird problem. Whenever I do a MidiPrep with Qiagen columns I end up with very little DNA even though my o/n culture had the right OD and looked nice

I have done everything according to the protocol, used the right buffers, waited the right amount of time etc. But still-very little DNA.

I have discarded my Ethanol and my Isopropanol, still it didn't work properly.

Any suggestion on what to do??? I mean, c'mon, using these columns is just the simplest thing there is!!! It is seriously bugging the **** out of me.

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I had sort of the same problem lately. Turned out that our kit was old and not stored properly. So you should check that. If that's ok: do you probably have low copy plasmids? Or have you asked somebody to do the same midi prep with your buffers? Sometimes when you get too used to a certain (boring) protocol, mistakes start creeping in...
Oh and: is the product that you produce with the midi ok? Done a restriction digest?

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Hi,
sorry for my question but... Are you sure that the columns are available for the length of plasmid?
The columns are plasmid lenght dependent.

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It basically is a "standard length" plasmid. Nothing special, about 5kb in size. As for the other questions, the kit is brand new and somehow it worked with 2 people in the lab whereas one of my colleagues and I are just bummed out And we both don't have low copy plasmids.

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then I suggest a thing most evil....

Midiprep old fashion way!

Rather then using column binding, centrifuge the DNA out... along with some protein... which has to be removed usually by phenol-chlorofrom

EDIT: Alternatively you could ask the two colleagues of yours to land a hand and conduct your midiprep. They must be doing something right and should produce your plasmid.

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Guess what I did




Yeah, true. But actually, I should also be able to do a midiprep according to a standard protocol. And since I have to do Midis from time to time, I'd sooo like to know what I'm doing wrong, which is apparently nothing cause a senior technician in our lab did the procedure with me and we couldn't find the mistake...it's just plain spooky...I'll try again on the weekend

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I think your bacteria are spitting out your plasmid, or are not at high copy number. Make sure that transformed cells are fresh or if you are going from glycerol stock, re-streak and grow from a single large colony. You might also want to re-make selection agent to ensure that non-transformed cells are not viable.


cheers.

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Hey britzelbeere:
Don't feel bad - I am still trying to get a "simple" ligation and transformation going. We are a new lab and apparently the law of the cosmos is that nothing works at first in a new lab...grrr...
Anyway, sometimes the preps can be a pain! In addition to what everyone else is suggesting, try these ideas:
1) even though your prep is growing to a certain OD, try to let it grow a little longer. I have done this with some tricky plasmids and it really helped.
2) take the Qiagen columns and rap them against a counter a couple of times. This is not to release your anger , but to loosen up the resin in the column (sometimes it is packed a little too tightly).
3) it's a total drag, but try taking the aliquots that Qiagen recommends at certain steps: maybe you can see a pattern;
Good luck! Keep your chin up - it'll probably resolve itself and then you can be left wondering what the h*** happened
mito1

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what type of E-coli you are using? some types are very bad for getting the plasmid.

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