maxiprep troubleshoot - (Feb/12/2007 )
Hi, I cloned my gene of interest into pCRII vector and did miniprep.I selected the positive one from miniprep and proceeded to maxiprep.The growth of the cells is good. But for some reason, the maxiprep is not producing any DNA pellet at all. I dont know the reason. Is it possible that after some time, the plasmid will come out of the bacteria?
Please help me
Yes, given enough time a plasmid will fall out of the bacteria. Although under such situations it is more likely to become hideously mutated, which bit deleted and swapped around. How long did you leave the cells? Where they kept in a refrigerator or out at room temperature?
As for losing the plasmid... how did you process your cells? Via column or via alkaline lysis?
If by alkaline lysis, was there a pellet after addition of isopropanol?
In the worst case senario, (ie all your cells are dead), you can retransform your miniprep DNA back into cells and conduct the maxiprep from there.
Agree with perseneblue.
Plus, have you grown your cells with any antibiotics? If not, the cells may have kicked out the plasmid.
Are you using the Qiagen kit? If so, make sure you mix thoroughly after the addition of isopropanol (before the first spin) as incomplete mixing means the DNA won't precipitate out...
As for losing the plasmid... how did you process your cells? Via column or via alkaline lysis?
If by alkaline lysis, was there a pellet after addition of isopropanol?
In the worst case senario, (ie all your cells are dead), you can retransform your miniprep DNA back into cells and conduct the maxiprep from there.
I left the cells at 4 C and also I had glycerolstock.I used both of them to regrow. The growth was ok but no DNA on isolation.I used the column and there was no pellet after isopropanol precipitation.
Plus, have you grown your cells with any antibiotics? If not, the cells may have kicked out the plasmid.
I grew them with Ampicillin.
I used QIAGEN and I never had this problem. I mixed thoroughly after addingf isopropanol.
have you checked if all the buffers in the maxikit are fine.
Did anyone else in the lab also have a similar problem?
Did anyone else in the lab also have a similar problem?
I agree. Do check. Also check if the expiry date on the columns have not passed.
The cells should have been okay if kept at 4 Celsius.
Do you still have the miniprep DNA. It is now your backup if all else should fail
Now I have same problem, growth is ok but very poor yield of plasmid dna via maxi midi, mini, etc.
I tried different commercial kits (sigma qiagen, genscript) and with all same results.
Plasmid is there but in very low concentration.
other construicts that I worked in parallel are ok.. yield is ok, but with this particular one (15000 kb) seems that its spit out of cells if overgrown. Today i did maxi from 250 ml OD 0.45 and pellet.. again .. pooor.
dont know what happen. when i got my clone i could do minis normally (although with phenol chloroform) and now.. blaaa. some times i get apparently contamination of a smaller plasmid.
if anyone has sugestions... i would appreciate it..
thanks
ps.. ampi ok, LB media, strains.. just getting transformants with jm109. some slow growing xliblue and non for top10, dh5alpha, dam negative NEB (commercial).