23 replies to this topic

[MaggieRoara]

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

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.

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

[shimshady]

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.

Attached Thumbnails

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[MaggieRoara]

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

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.

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

[shimshady]

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.

[phage434]

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.

[MaggieRoara]

shimshady, on Mar 12 2009, 06:30 PM, said:

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.

So you are saying that you want to digest a large amount of DNA and extract a large amount of the digested DNA from the gel? what are you going to do with the DNA from gel extraction?

[shimshady]

I just want to have alot of DNA, and concentrated.  I bought these clonables novagen dna ligase master mix which requires 5 ul of plasmid and insert total and 5 ul of master mix.  So i need concentrated plasmid for this (50ng/ul or so).

[MaggieRoara]

shimshady, on Mar 16 2009, 05:33 AM, said:

I just want to have alot of DNA, and concentrated.  I bought these clonables novagen dna ligase master mix which requires 5 ul of plasmid and insert total and 5 ul of master mix.  So i need concentrated plasmid for this (50ng/ul or so).

you should do a maxiprep... given large amounts of clean DNA... i recommend qiagen's maxi prep kit

[shimshady]

Just wanted to let you all know that i got the ligation to work out.  Thanks for all your help!