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Low plasmid yield after Miniprep, DNA in the first wash flow through (PB)

miniprep binding buffer low yield

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#1 Olesia

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Posted 14 November 2014 - 07:27 AM

Hi,

 

I've done mutagenesis on a couple of different plasmids and now I'm trying to get a decent amount of plasmid, using a miniprep, to send it for sequencing to see if the mutagenesis worked before I go onto doing maxi. The problem is that I get really low plasmid concentrations (ca. 10ng/ul!) irrespective of the volume of culture that I use (tried from 2, 5, 10 and even 30ml), plasmid type (pLightSwitch / pcDNA) or E.coli strain (Top10 / XL1 blue)... I noticed however that when I was repeating my experiment and checking the flow through after each step, that DNA concentration was very high after the first wash step with PB buffer... in my last plasmid extraction attempt I included 'control' samples - 5 ml of the same O/N culture eluted after using either PB/PE wash and 2x PE wash. I got from 2 to 5 times more plasmid when doing 2x PE washes instead of using PB in the first one. Has anyone had an issue like that before? I am a bit worried about the purity of my sample (the ratios were good though) but I guess the main point of PB is to improve DNA binding and PE is the wash buffer that should remove all the contaminants? 

 

Would appreciate any advice smile.png! (I did manage to get enough DNA for my sequencing but I still don't understand the reason behind it all and it's really bothering me! Please help!)

Thank you!



#2 miST32

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Posted 14 November 2014 - 08:58 AM

That does sound weird.

1.  I would check the pH of the buffer.  If it isn't around 7.5, you may have problems promoting DNA binding to the column.

I also considered propanol concentration in PB, but I kind of doubt it because one can wash the column with 35% Gu-HCl (no alcohol) to remove short oligos/primers, and have little impact on yield in my experience.  It's odd that PB is eluting your DNA, and it makes me suspect the pH first.

2.  If you precipitate your PB-eluted DNA, what does it look like on a gel?  Are you recovering degraded pieces of DNA perhaps?  These might plausibly elute in PB is sufficiently short, I think.  I'd still suspect the pH first, but could be wrong.

My understanding is that PB denatures contaminating proteins, whereas PE washes away salts (such as the chaotropic guanidinium in PB).

**Edit:  My colleague also suggested checking the other buffers involved in your resuspension/lysis process - perhaps the lysate is altering pH or other property that reduces binding in PB buffer?

 


Edited by miST32, 14 November 2014 - 09:10 AM.






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