What does this look like to you? - (Mar/06/2009 )

shimshady on Mar 10 2009, 05:15 PM said:

From the picture, I can see that your wells were "glowing". I think this might be because your DNA was dirty. I am assuming you are using DNA from miniprep. Perhaps you should review your Miniprep methods. Or if you wanna be extra sure, do a maxiprep, and check your purity readings.

Like i said before do a virtual digest with the NEB cutter, they give you a "gel picture" if it corresponds to your gel then good. Load less of the reaction or lower the light settings for your gel pics as well, they are too bright, sometimes if you have similiar sized bads, they might glow so much, they appear as one band.


Do send me a sequence of your plasmid, and i can show you how to run it through the virtual cutter. May I also ask what your rational is behind digesting one enzyme by one, is there a problem with running a double digest?


And i dont think the shaking has anything to do with the reaction.

I did not add water to the reaction as the plasmid was already in about 27-28uL of nuclease free water. Adding everything else would have brought it to around 30uL.

So i did the NEB Cutter and the base pairs between the two digestions is 12 bps. So i should see something around 5.6 kb. This is also the reason im doing a sequential digestion, due to the fact that they are so close together. How do you check for purity, other then running a gel of this? Thanks

shimshady on Mar 11 2009, 01:15 AM said:

What's the final volume of your digestion reaction? Asuming Buffer 4 is at 10x you are not adding the right amount. 30ul DNA + 1ul Exzyme + ?ul BSA is > 30ul total, which will require 3ul buffer, so I think you might need to adjust this.

Hope this helps.

It was really 27.5 uL DNA, i was just rounding. So it would have been 32uL total in the reaction with .5 uL BSA

That is a very concentrated DNA reaction. Normally, in 30 ul I would react 200 ng of DNA. The nominal reaction is 1 ug in 50 ul. This is 5x more concentrated. I'd recommend scaling back the amount of DNA. Using large amounts of DNA and little or no water is in general a bad strategy, as the DNA is concentrated (with columns or precipitation, or whatever) in combination with undesired contaminants, such as ethanol. Diluting the DNA also dilutes these contaminants, and can save you from many problems. If I wanted to digest this much DNA, I would do it in a 300 ul reaction, not in a 30 ul reaction.

phage434 on Mar 11 2009, 05:59 AM said:

go ahead and dilute the DNA.... gels should be able to detect up to ng levels of DNA so dont worry. Use the NEB cutter and choose other enzymes since the interval between the two sites is too short

Here are pics of the gel previously and the new gel pics with new dna (they are labelled). The top gel is only one digestion. The second is both digestions. The bottom is previous gel. it looks digested to me, its higher in bp's, which seems to me that the undigested dna is mostly supercoiled. I think the previous gel is mostly digested but maybe the gel is overloaded, what do you think. I think it worked.

Thanks for your help.

shimshady on Mar 12 2009, 07:39 AM said:

the pic in the middle is good!... i think you got it. If you wanna be extra picky, you can try like i said with other enzymes with more far apart cut sites, but this is fine!!!

yeah you did it

Thanks for your help, but i really want to be able to do this with a large amount of DNA, say 3ug. The gel purification doesnt give that good of return on my product, like 50%. I want more since i have to use for quantitation and digestion and i want to make it really concentrated product as well. My question is how do you compromise the volume of the digestion and running the gel to purify it. You get a huge band that requires alot of gel in return requires alot of binding buffer for binding to the column. Do you understand what im saying. Your help has been much appreciated, kept my from pulling my hair out, lets hope the ligation goes well.

It is rare that you need a highly concentrated product. A good place to be operating for ligations is around 10 ng/ul, which is easy to achieve with dilute DNA digestions and normal elution from columns. Higher DNA concentrations favor concatenation of vector rather than recircularization.