4 replies to this topic

[MyProteinBulliedMe]

Ok so here's the whole problem. My objective is seriously simple: I'm trying to purify the linearized DNA from restriction digest with a HindIII single cut (NEB). So far I've been using DNA purification kits form three different companies gave smear (Qiagen, Zymo, Promega). All are new kits. All other purification using these kits has been good (5kbp ones from digests, 400bp ones fro PCR), just this particular 10kbp DNA gave smear. I don't think this is Kit issue.

I also tried eluting with water, TE, and with EB buffer that came from our Qiagen Kit. All resulted in smear. I've checked DNA+binding buffers and it resulted in single band and not smear. So something happened in the silica or whatever the white paperish stuff in the middle of column is made of.

I've also thought about the DNase. I know Proteinase K remove most DNase, so I've tried putting ~1.5ul Proteinase K (Roche) in ~25ul reaction, incubated 20 minutes at 55C. When I ran it immediately on gel, it gave single band. Tried purifying it using Kit eluting with EB buffer, it gave smear.

I also tried phenol chloroform because this method kills most of DNases, and ethanol precipitated it overnight. The next day I resuspended it in water. It gave single 10kbp band (YESS!!!). Then I stored it in -20C for a day. The next day I ran it on gel and there is smear. (This is why I thought freeze thaw caused smearing).

The reason I'm eluting with water is because I'm using it for assays that is quite sensitive to Tris/EDTA besides I'm using it immediately within 24 hours (so it's not going to be stored for a long time). But now I don't care anymore I just want my plasmid intact.

So now I'm seriously running out of option...it's just a 10kbp linear DNA how the hell it's degraded THAT fast (<1 minute) in both EB buffer, TE buffer, or H2O?

I used different pipet and different tips, even opened new ones. I tried with different loading dye and tried company's loading dye. I made new gels. I washed the gel tray in 0.2M HCl and ethanol and rinsed them good with water. I post-stained in case EtBr get in between the linear DNA and screw it up. I gave offerings to the DNA gods. My Restriction Digested DNA is now still intact 10kbp band while everything that I've purified out of it is a smear. Now I'm trying to add more phenol chloroform steps hoping to kill whatever is degrading my DNA.

Any help appreciated I'm confused like hell

EDIT: I'm attaching the gel picture so you guys can see the smear. Single cut is HindIII to make it linear, double cut is HindIII with another single cutter to check (the second band is faint but you can see it's there), and the rest are smears.

How many freeze thaw does it take to degrade linear DNA that is stored in npH2O?

Suppose I have a 12 kbp pure linear plasmid that is in nanopure pH 7 H2O with concentration of about 1 microgram per microliter. Then I freeze thaw it three times (from -20C to RT). Will this make >95% of it degrade into pieces of 500bp-8kbp smear where brightest around ~3kbp and least bright at 500bp and 8kbp?

Attached Thumbnails

• 

Edited by MyProteinBulliedMe, 26 April 2011 - 11:46 PM.

[phage434]

No, probably not.  You likely have DNAse contamination or the pH is much lower than you think.  You should be storing your DNA in TE.

[MyProteinBulliedMe]

I checked the pH of my water, and it's 7 (used 2 methods: pH machine, paper test). It's coming straight out of our nanopure machine which is used for DNA sequencing, ChIP, Bisulfite, etc.

Edited by MyProteinBulliedMe, 26 April 2011 - 11:47 PM.

[phage434]

What you are measuring is the pH of the paper or the instrument.  The water is so pure that any minor contaminant will overwhelm the pH of the water and set the pH of the solution.  This is why you buffer solutions.

[mole]

Hello,

Hope i can help you.

So please let me know If have PCR Purification kit available in your lab if yes from which brand ?

Mole .