Do you have any problem with QIAGEN Plasmid Maxi kit - (Jan/29/2006 )
Hi dear All,
When I use the QIAGEN Plasmid Maxi kit to prep the DNA for transfection, I cannot get DNA pellet. I repeated three times and just can not get my DNA. In the 3rd time I retransformed my plasmid and Pick a single colony and culture in 2–5 ml LB medium containing Ampicilian, incubating for approximately 6 h at 37°C with vigorous shaking and than add 1ml to 250ml LB, shake overnight(16-18hr).
I finish the plasmid prep. accoding to the protocol, but I just can not get the DNA, I feel very depressed. My boss will also be mad with me.
Does anybody have the same problem and can you give me some suggestion?
I have used the kit many times and it is quite good but there are a few niggly bits. The main problem is loss of the pellet after precipitation. When you precipitate your DNA, are you spinning in a 25 ml centrifuge tube? I found that it is difficult to see the pellet and is very easy to lose when you decant the supernatant. Also, its hard to resuspend the pellet in a volume less than 500 ul - 1 ml. Here's what I do, I split the precipitated solution containing the DNA into 13 X 2 ml eppendorf tubes and centrifuge to collect the DNA. I then remove the supernatant very carefully using a 100 ul pipette tip. It's hard work but using a 1 ml pipette tip will amost surely gurantee that the pellet will dislodge and be thrown away with the supernatant. After washing the pellet with 70 % ethanol, I air dry the pellets and then resuspend in 200 ul of water. I transfer the water from tube to tube, concentrating it with DNA. You will see that the solution becomes viscous the more concentrated it becomes.
An alternative method is to precipitate the DNA with ethanol rather than isopropanol. This will make the DNa more visible and easier to prevent its loss.
Hope this helps.
Indeed, loss of your DNA after isopropanol-step is the most 'risky' part of you prep.
so be carefull when decanting Supernatant, or pipet it away and let just a bit of it in the tube and then add more 70% EtOH. Your total % of alcohol (EtOH + Isopropanol) is still plenty for your DNA to remain peletted.
i always precipitate DNA eluted from the column with iPrOH and centrifuge immediately 13000 rpm 4C/30min in Corex tubes
then i dry the pellet and resuspend it in tris-EDTA for further precipitation with sodium acetate and EtOH 100% in Eppendorf tubes and then washings with EtOH 70%
finally, the pellet is clean and resuspended in water or Tris-EDTA
sometimes you might not be getting the pellet because you might be amplifying the plasmid in a low-copy vector. I normally pick a colony and add it to 25 ml of luria broth (with antiboitic of choice) and let is shake overnight at 37. next day, i add 5 ml of this to a 500 ml of luria broth and follow the OD. When Od reached 0.6-0.8 I add chloromphenicol and let it shake fro an additional 12-15 h. This is what I purify. The pellet that I get is very mich visible. and i can dilute it in about 100 ul of TE buffer.
I agree that could be an issue with low copy number. The qiagen maxi prep is widely used so it is unlikely there is a problem with the system. Are you able to do minipreps with this plasmid?