Protocol Online logo
Top : Forum Archives: : Molecular Biology

Problems with Plasmid purification - no real precipitation of genomic DNA (Oct/26/2006 )

we use routinely Qiagen plasmid purification kits, and starts as follows

1. resuspension of bacteria pellets in P1 (Tris/EDTA + RNAse)

2. lysis (200 mM NaOH, 1% SDS)

3. Neutralization (3 M K-acetat)



we get rather a slimy viscous solution rather than really precipitates; slime is not to precipitate even with high speed centrif├║gation (may be ultracentrifugation will work)

I must admit until today the kit was really reliable and reproducible


The only thing which was strange that the provided K-acetat solution was yellowish as normally it is not; i don┬┤t know the reason for this

We still have the viscous solutions, and like to know what to add to precipitate the genomic DNA and cell debris; plasmid should remain in solution

-The Bearer-

basic questions i should ask first :
do you wait 10-15' between addition of so 2 and Sol3 ?
are they fresh solution / kit?

after that, i'm wondering why the solution 3 is yellow. I would suggest to prepare a new one.
But if the solution itself is viscous, that probably means you have too much bacterias for the volume of solution used.

-fred_33-

I agree with fred regarding the amount of bacteria. You should be able to understand whether you have too many bacteria after you add solution 2. At this time point, your lysate should really appear gelatinous if you have too many bacteria... You can prepare the solution 3 but if I were to be you, I would also contact Qiagen and ask them to substitute the neutralization solution

-dnafactory-

I would say something is not right with the neutralisation buffer. did u try adding more of it . This may help. But I would suggest to get a new one or prepare a fresh one.

-scolix-

i had the same smears on gel after miniprep. I used a promega kit which has RNase included in the Cell Resuspension reagent. I wonder what caused those smears, that made my bands not clear

-arvinsign-

I use the same Qiagen kit in my lab and if Buffer N3 (neutralization) is yellow I would say that is where your problem lies. After you added it to the solution did you get the normal whiteish snot-pellet like separation?
If you want to prepare a new solution, try this website (http://130.15.90.245/wormlab_recipe_book.htm#Commonlab) or you can order another bottle of the buffer from Qiagen.

I agree with Fred on the possibility that you may have too many cells for the solution, but the kit specifically says to let the lysis reaction go no longer than 5 min. Having left this step go too long before I know that can really mess things up later on. Best of luck!

-Montana81-

i agree with Montana81
i think the problem is definitely your sol3. i am using the same kit and after addition of sol 2, you get viscous solutions. neutralization buffer precipitates everything in the solution other than plasmid DNA. it gives you a cloudy solution. did you see a cloudy soln after the addition of sol3?
if the answer is yes, then your soln3 is fine, i guess. (i never see a yellow neutralization buffer since it is simply a K-acetate soln).

-dodosko-

QUOTE (dodosko @ Oct 26 2006, 05:53 PM)
i agree with Montana81
i think the problem is definitely your sol3. i am using the same kit and after addition of sol 2, you get viscous solutions. neutralization buffer precipitates everything in the solution other than plasmid DNA. it gives you a cloudy solution. did you see a cloudy soln after the addition of sol3?
if the answer is yes, then your soln3 is fine, i guess. (i never see a yellow neutralization buffer since it is simply a K-acetate soln).


thanks to all contributors;

in the meantime I spoke to the manufacturer who admit that solution 3 may become yellowish if not light protected but this should not affect the preparation:

when adding more solution 3, we got only some small fluffy clouds but not as big as usual, and solution is still viscous

additional contributions are still highly welcome, especially how to separate plasmid from genomic DNA in our case

-The Bearer-

Well since Qiagen says yellow is an acceptable color, is it possible after adding Buffer 3 to transfer out the bottom viscous layer (leaving the white portion) and repeat with another round of Buffer3?
Ultracentrifugation may work but I would be worried about pulling the plasmid down along with the genomic DNA in that case. Anyone else have a suggestion?

-Montana81-