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kiki06 09-08-2006 07:49 PM

Very low plasmid yields from mini-prep
 
I am currently getting very low plasmid yields form mini-prep after growing e.coli bacteria overnite.

What may I be doing wrong?

thanks
Kiki :)

kmunson779 09-08-2006 09:15 PM

Re: Very low plasmid yields from mini-prep
 
Lots of things could be going wrong.

Could you post more details, like your protocol and/or what kit you're using?

kiki06 09-11-2006 06:38 PM

Re: Very low plasmid yields from mini-prep
 
Hey Kmunson!,
I used Qiagen kit and grew up E.Coli bacteria overnite.

I have had this before and it seems to occur with certain plasmids only.

thanks
Kiki

kmunson779 09-11-2006 07:05 PM

Re: Very low plasmid yields from mini-prep
 
For the Qiagen spin miniprep kit, here are a few things that I've found helps yield.

1) Don't use too much O/N culture. Although I've never run a side-by-side comparison, I tend to get better yields using 5-6 mL rather than 7 or 8. More is not always better!

2) If your kit has LyseBlue, use it. When I started adding this reagent to my preps, I noticed that my usual way of handling samples didn't always make the color turn the way it should. To be precise, I tended to mix too few times after the neutralization buffer addition step.

3) Load your sample through the column twice. This may be my own little paranoia, but I like to run the lysate through twice (take what went through the column into the collection tube and reload it into the column) and the elution through twice. I don't think it hurts anything, and I figure it gives molecules one more chance to bind (or elute).

4) Let the wash evaporate completely. I've noticed, even spinning an extra minute to get rid of the wash buffer, I can often still smell the ethanol. Now I like to let the columns sit and air dry in the elution tubes for a couple of minutes before I add the elution buffer.

5) Heat the elution buffer. One I've never tried, but it's suggested in the protocol (I think) -- Heat the elution buffer to 65 degrees before adding it to the column (and I might incubate the column with elution buffer in a heat block for a minute as well) -- the heated buffer is supposed to be better at eluting DNA.

6) Don't throw away the column. I hold onto the columns while I spec my first elutions -- if a prep has a low concentration, I run another 50-100 uL elution buffer through the column and check its concentration. If both elutions have some small amount of DNA, I pool them, ethanol precipate, and resuspend in a smaller volume (say 10 or 30 uL). This is some extra time, but I think it's easier than trying to redo the entire miniprep.


Remember that yield for these kits is based on two factors: how much plasmid you put in at the beginning, and how much makes it through the purification process. I've talked about the second factor, here are some pointers on the first:
1) Check to see whether the plasmids that aren't prepping well are low copy number.
2) Keep an eye on the size of your bacterial pellet. Maybe you don't have as many bacteria to begin with.
3) Associated with both factors: I seem to see an inverse correlation between the size of the plasmid I'm purifying and the yield: I think larger plasmids (>10 kb) don't make it through the purification process quite as well (perhaps if they get nicked they get caught up with genomic DNA during the lysate clearing step? and/or they don't elute as well from the column due to their increased interaction area?), and I suspect they will have fewer copies per bacterium as well (though I don't have hard data on that).

Well, that's my $.02 (or was it more like $.25?)...

kiki06 09-13-2006 06:51 AM

Re: Very low plasmid yields from mini-prep
 
Wow!
thanks for a great response... I followed your protocols lets see tomorrow :)

Thanks
Kiki

hsip_15 11-19-2006 01:12 PM

Re: Very low plasmid yields from mini-prep
 
Haik,

I have been getting low plasmid yields as well. My plasmid is suspected to be a low copy plasmid since the size is about 22kb through literature.

However, I have tried heating the buffer and re-eluting the columns.

Heating the buffer seems to have not much effect as I get the same consistent faint bands in electrophoresis. Does anyone knows why the elution buffer is recommended to be heated up to 70C before eluting the plasmids?

Then, electrophoresis for the re-eluted columns, did yield a faint band in electrophoresis as well.

Instead of modifying the Miniprep techniques, what can I do to my bacteria in order to produce more plasmids if it is a low copy plasmid? In the Qiagen kit, it is also stated that in order to yield higher amounts of plasmids, the NaCl amount is doubled in the LB medium. Why?

Thanks.

songsong 05-14-2007 12:33 AM

Re: Very low plasmid yields from mini-prep
 
What is the lyseblue,can it help lysis?

kmunson779 05-14-2007 02:11 PM

Re: Very low plasmid yields from mini-prep
 
LyseBlue is a reagent in the Qiagen kits... basically it's just a pH indicator that turns blue when the solution is alkaline (during the alkaline lysis step).

It has no effect on lysis (it's only been included in the kits recently), but it's a good visual indicator of how well you're mixing your samples.

songsong 05-15-2007 07:51 PM

Re: Very low plasmid yields from mini-prep
 
thanks, I have found one and used it. Just like what you've said it make me noticed that my usual way of treat sample didn't work very well.

thebetafemale 01-19-2012 03:48 PM

Re: Very low plasmid yields from mini-prep
 
Curious. I read on another one of these forums/wikis (Open Wet Ware I believe) that doing two PE washes with the Qiagen kit gets rid of extra reagents that might later interfere with sequencing. I tried this today because I've been having trouble with getting good sequences of my plasmids.

But, my yields which have been sort of low (100-150ng/uL in 40uL of buffer) were even lower! Around 90ng/uL in 35uL. I was definitely expecting over 200ng/uL which is what I need for sequencing. The whole reason I did this miniprep today was to try to get a higher concentration for sequencing, and it was a total waste of time.

Do you think that the extra PE wash step washed away a lot of my DNA and that I should not have done it?

Other things that were different about this particular miniprep: I started with 5mL cultures instead of the usual 3 because I thought that more bacteria = more plasmid. Also, the plate I pulled them from had been sitting in the 4 degree refrigerator for about 5 days so maybe they kicked out the vector? It's a big plasmid - 7600kb including the insert and cloning has been difficult in general.


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