problems with yields from qiagen spin mini prep kit - (Jan/25/2007 )
hi all,
our lab has been having major problems with yields from the qiagen spin miniprep kit. the cells we are using are TOP10 from invitrogen. the yields we are getting are as low as 20ng! has any one else had this problem? any ideas?
our lab has been having major problems with yields from the qiagen spin miniprep kit. the cells we are using are TOP10 from invitrogen. the yields we are getting are as low as 20ng! has any one else had this problem? any ideas?
Something similar happened to me in the past. One first step is to check if you get enough bacteria. Do with spectophotometer and compare with the values they give. What I do now is doing 20ml of bacteria culture instead of 5ml and it works better. You do exactly the same way (using more reagents if required, double should be enough) and use the same spin column all the time. Your final concentration should be much higher.
If you try just let us know if it worked for you.
thank you!
il try it as soon as i can and get back to you! its really wrecking our heads!
Try using another Qiagen miniprep kit - either buy another one or use someone elses. Then you'll be able to determine whether it is the kit or your bacteria/plasmid/your procedure that is mucking you up. I also use TOP10 cells and the Qiagen miniprep kit and have never had any problems with either. Definitely interested in the answer when you get it.
I had the same problem so you could try this:
1.check the quatity of cells and use the appropiate kit for it (some times people think that the have fewer cells and use the mini and the membrane get block with all that material)
2. check the viscousity of soln when adding the EtOH if too viscous add more EtOH
3.add the aditional cleaning step that they mention in the protocol
4.dry well the membrane centrifuge 1-2 min
5.add 1/2 elution buffer let stand 5 min, centrifuge add the last half again let stand 5 min and centrifuge.
hi! thanks for the reply. there are five lab members ranging from senior to junior in our lab and we all have the problem! we have bought in loads of new kits so i dont think its a problem of contamination or faultly kit.
No one has mentioned that this can be highly dependent on the plasmid you are purifying. Low copy plasmids will give low yields. Also, if selection is poorly maintained during growth, many cells fail to have plasmids, and this can dramatically reduce the plasmid yield. Common problems are failing to add EtOH to the wash buffer, or using wash buffer which has reduced EtOH concentration from evaporation. There is a diagnostic you can run described in the protocol which saves small amounts of the lysate, flow through, wash, and elution, and runs them on a gel. You may get insight from running these samples.
We faced similar problems and it was because the columns were too old!(In years!) Is that the case here?
il try the diagnostic test you mentioned in the protocol. i have checked many times that to make sure TOP10 is a high copy plasmid, it is so its not that. also EtOH is always added! we cant all be forgetting it! the evaporation issue is valid and i will make sure all lids are tight and opened only when needed.
thanks, il let you know how i get on!
hi! we have just bought in brand new ones and same story so i doubt it. unless the supplier is giving us old stocks? i dont know if thats possible though