plasmid prep - help (Apr/01/2008 )

Hi everybody,

I have problems with preping a plasmid I made (~ 10kb). It`s a luciferase construct containing high repetitive sequences (problems with unwanted recombination, too). Maybe that`s why I am still not able to prep it. The plasmid is going to be used for transfection/luciferase assay, so I need good quality/quantity DNA.

I did mini prep from a 2ml ON-culture with homemade buffers (alkaline lysis). I have got some DNA but the quality/quantity is just not good enough.

I tried midi prep (50 ml ON culture) with Jetstar2 kit (columns), Qiagen midi kit, Promega midi kit. No no no DNA at all. I also divided the culture into two just in case if I would overload the colmuns. The same result. I warmed up the elution buffer, the same result.

I tried midi prep with PEG8000 (no columns), Phenol-chloroform extraction (no columns). Almost nothing and bad OD ratios.

I tried miniprep 2 and 5 ml ON culture (from a single colony) with EZNA miniprep kit and Qiagen miniprep kit. No no no DNA.

I combined minipreps (~12 pieces) and tried to purify with Phenol-Chloroform and gelextraction columns. Nothing, nothing (I eluted both in MQ water and 0.1x TE pH8).

I combined minipreps the same way but used Qiagen gelextraction beads. That worked somehow (at least I saw some DNA on the agarose gel) but I think the beads affect the ODs (measured with NanoDrop). Using that DNA, I tried the luciferase assay but I got different results every time and the luciferase activity is quite low (I suggest the DNA concentration is lower than that I determined by NanoDrop).

So, if you have any tips or ideas, please share with me.

Thanx in advance

Try growing bacteria at 30°C and grow them for a longer time, 4-5 hours extra should increase yield and maintain repetitie sequences (I grow retrovirus with 2 LTR's at 30°C and the plasmids are roughly 15 kb, get nice yields with Qiagen miniprep, think other methods should work too). Only thing that comes to mind to pay attention to is to make sure you have elution buffer (TE or Tris) on there a little longer maybe.

Thanks for the reply.

I grow them at 30 degree (sorry, forgot to mention )

I also have tried that . It did not help. I thought first that it get stuck in the column but I did not get DNA from the prep without columns, either

Really, I don't know what the problem is or what else I could try. I tried SURE, DH5 alpha, DH10B, NM522, JM109 bacteria, as well (against unwanted recombination and just in case it would help for the prep).

Anyway, thanks a lot

Hey Vairus,

what kind of bacteria do you use? What kind of kit do you use for big preps? Would a maxiprep help??

Make sure you have a high enough culture OD before you do your extraction. The kits you are talking about should list the recommended OD. I usually grow mine for 12hrs in 5mL media from a single colony

Hey,

thanks.

I grow the mini cultures for about 16h and the midi cultures from a big piece of frozen stock for 16-20h at 30 degree (shaking at around 235rpm). Mini cultures are in 13ml round-bottomed tubes, midi cultures in 500ml glass flasks. The cultures look quite cloudy before I harvest bacteria.

Quite cloudy and actually cloudy are two different things. I'd check the OD just to be sure

Hi,
I had the same problem before. To solve this problem, you may try the following.

Miniprep the plasmid.
Re-transformation into e. coli. Optimize the condition to obtain maximal number of colonies.
Transfer all the colonies into suitable volume of LB(with selection drug)
Incubation until desired OD is reached.

Hi,

just want to update the prep story.

I grew a maxi culture (500 ml) and followed exactly the 'alkaline lysis, SDS' protocol in Sambrook (Molecular Cloning 1 protocol 3). Then the 'PEG8000 precipitation' protocol in the same book (protocol 8 or 9, don't remember the exact number) was followed. The modifications are that P:C:I extraction was repeated 3 times, no chloroform extraction and the DNA pellet was resuspended in 50 ul. I got around 100ug DNA with good 260/230 and 260/280 ratios. It is not much DNA for maxiprep but enough for transfection.

It took me 2 days to do it but worth after so many failed prep as I mentioned in the beginning. I remember that we did maxi preps without columns and kits in "basic molecular biology" lab course at the uni (long time ago). However, when I started to work in a "real" lab, there was/is only kits (easier, faster, more efficient etc.). I think I have learnt a lesson.