low maxi-prep yield with qiagen kit - (Apr/19/2007 )
I've done a maxi prep using a Qiagen plasmid maxi kit, but had a very low yield. Not even 100micrograms total, when the purification column is suposed to bind up to 500micrograms of dna.
At first I thought I had lost some of the pellet when I did the isopropanol and ethanol precipitations, but apparently that wasn't the case. I took the samples they suggest at various points in the protocol and the outcome was this:
1st sample - after lysis and centrifugation of cell debris - good amount of plasmid dna (as expected)
2nd sample - flow-through after passing the previous solution through the column - no dna present (good)
3rd sample - flow-through after washing the column - no dna present (good)
4th sample - flow-through with the elution buffer - very low amount of dna when compared to the 1st sample (when it should be approximatly the same, right?
From this I suppose the problem was that the plasmid wasn't properly eluted with the buffer, since there was no dna in all the other solutions, right?
What should I do to improve this? Pass the elution buffer 2 ou 3 times through the column?
Has this happened to anybody else? If so please let me know how did you work things out.
well i use twice the culture volume as they recommend.
second way to increase binding is to use the chloramphenicol method to decrease amounts of RNA.
finally eluting twice gave me little more plasmid.
In general, the quiagen preps didn't gave me more than 250-300µg plasmid. For a 13kb plasmid, i only got 90µg:(...
You should also warm the elution buffer, make sure you are using one with pH 7.5 or above, and let it sit on the column for 5-10 minutes. Eluting more than once and pooling the result will also help.
Make sure that your elution buffer is good? DNA resting in column so long before eluted or the elution rate quite fast? Repeat elution with elutant twice or more, pass slowly buffer (in my case, i use a syringe disposed upon the column, which allows buffer fall down drop by drop).
Grow up more bacteria that what they suggest in the book.
Oh, and really, REALLY,make sure the cells are properly lysed before dong anything else.
I checked the buffer pH and it was 8.4, which is ok, so I guess I'll try what you guys suggested.
Thanks for the fast replies
Is your plasmid a high copy or low copy? This can make a difference.
Also try to grow a 10 ml day culture and use it to inoculate a 250 or 500 ml overnigth culture. This should help if plasmid is lowcopy.
Yep, it's high copy...
Well, I won't be doing the maxi prep again so soon after all cuz the amount of dna I got was enough for a couple of weeks, but eventually I'll give it a try again and let's hope it works...
Thanks for the feedback everyone