Ligation

[Deitiker, Philip R]

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of ChenHA
Sent: Thursday, June 28, 2007 9:12 AM
To: methods from magpie.bio.indiana.edu
Subject: Re: Ligation

Deitiker, Philip R wrote:

> 
> Somewhat new problem. 
> I am using Promega's Midiprep kit, it appears even after attempts to 
> dry off the ethanol the resulting DNA has EtOH, or at least, does not 
> freeze and has the trace smell of Ethanol. Last time I did an EtOH 
> precipitation on the midiprep DNA, problem is that much of the precipitated material did not resolubilize, possibly because I incubated it too long at 65'C I am weary of doing this again so we were really careful to dry the membrane, but I still recovered a great amount of DNA. Has anyone used the
Promega Midiprep kit and had similar problems?
> 
> Also, apparently the tube cultures of bacteria seem to work fine, but 
> a similar volume of flask culture seems to overwhelm the kit. Too much DNA? Is it possible to clog the DNA binding membrane with too much DNA?
"
I haven't use the Promega kit for a long time, but I think most kits work on the same principle.  I normally use Qiagen midiprep kit, although other companies do kits which work well.  I am puzzled as to why ethanol contamination should be a problem, since you should be able to see if it is properly
dried - if you can't see any liquid, then whatever ethanol that's left should be so small that it shouldn't affect most things you do.
"

Maybe you're right, there is one way to know simply do digestions
with both enzymes separately if they both cut once in a specified
amount of time. Anyway one has to repurify the DNA before ligation. 
Sounds good. 

My helper was concerned because the membrane clogged up on
one sample and we were forced to discard some of the lysate. 

"
 I normally just pipette the liquid out, leave it for a minute or two at room temperature, then pipette the remaining liquid again with a fine bore tip, (repeat a minute or two later if necessary if you can still see liquid in there), then resuspend the DNA in 50-70ul TE buffer after 5-10 minutes).
When the DNA is too dry, it makes it more difficult to resuspend.
"

I suspect that the insolubility of the Vector (BamH1 only) was the cause for the low level of religation/tranformation in the last experiment. Its been years since I have done phenol chloroform extraction and we worked with EtOH pellets so small you could barely see them. Needless to say getting DNA
into solution was not the problem.   The Vector BamH1/EcoR1 appeared slightly turbid but soluble, so that the problem with BamH1 does not explain its failure. It is apparent however I do need to get my efficiency up. 

"
  But if you get lots of dissolved DNA anyway, this is entirely academic, just spin down whatever that didn't dissolve.
" 

Typically I am retreaving 90ng/ul of DNA. 
The vector is 6300 bp so this should give about 2.5 fm/ul
[Who wanted fremptomoles? :^) ]

"
Wasn't there some issues with Promega kit? I think it's something about patents, so they had to change their kit components which didn't work well, but that was many years ago, I'd assume they fix whatever problems they had. (Who had the patent for the DNA prep and has the patent expired?)
"

Don't know. But thanks for the above, I took a look at my calculations
apparently a zero snuck into the calculations, I don't need >90ng/ul
of DNA which means I don't need to concentrate, Yeah! {wish Excel would not convert large numbers from Sci/Eng format to looooong numbers. At least use commas) The ligation should have worked anyway, but now I don't need to waste so much DNA.