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Miniprep-cell lysing problem

miniprep plasmid kit

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5 replies to this topic

#1 mysunking

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Posted 27 February 2012 - 02:01 PM

Hello all!

I have a question regarding the LYSIS step of plasmid mini-prep using a kit.

The problem happens after adding solution 3 (the neutralization buffer) to the cells. What I observed was a chunk of mucous in the solution. After spinning the tube at 12000-13000 rpm for 5 min (according to the protocol from the kit), the mucous does not precipitate nicely. It still kind of floating in the solution so made it very hard for me to take out the supernatant and to load it onto the column.

So what is this mucous after neutralizing the cells? Shouldn't I just see white precipitate (proteins) from the cell and shouldn't the white precipitate stick NICELY to the tube wall after spinning?

Please advise! Thank you for taking your time!

Best
M.Y.S

#2 HOYAJM

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Posted 27 February 2012 - 02:50 PM

Yes it should stick nicely to the tube wall after spinning. Try 13000 rpm for 10 minutes. That is what the Qiagen kit recommends. Maybe 5 minutes is not enough. Also, after you add the lysis buffer, immediately invert to mix. This will help give a more homogenous mixture and not such a clump. It says invert 4-6 times, but you should invert until it is completely mixed, just make sure you are gentle and it wont be a problem.

#3 mysunking

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Posted 27 February 2012 - 03:27 PM

Thanks Hoyajm!

I DO observe homogenous mixture after adding the lysis buffer...the problem happened after when I added the neutralization buffer...from some kits I observe nice white precipitates after initial spinning (13,000 rpm 5 min) but from other kits I observe this mucous after spinning. What could be the cause of this difference is what I am curious about.

#4 phage434

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Posted 27 February 2012 - 04:56 PM

What kits? I would guess that there are too many cells in your lysis prep. Perhaps you could try using less culture.

#5 mysunking

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Posted 27 February 2012 - 07:30 PM

It is the Biobasic E-Z10 plasmid DNA miniprep kit. I used overnight culture that was growing in 3ml LB+amp. I did the prep in the morning around 10, which is what I usually do.

#6 mysunking

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Posted 27 February 2012 - 08:53 PM

I should mention that it is very similar to the Fermentas kit.





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