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Digestion of clinical plasmids for posterior cloning


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#1 mjones

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Posted 06 January 2011 - 03:25 PM

Hi

I'm planning to digest a plasmid, extracted from a transformant E. coli using Promega MidiPreps kit, in order to bind the fragments to the PBK-CMV vector. Posteriorly, I want to electroporate and select the recombinant plasmid with my interest gene.

Now what I want to ask is, what do you think is the most proper reaction I must use to achieve this (in terms of plasmidic dna concentration, enzyme, enzyme units, incubation time and reaction volume)? How can I confirm that the reaction has been successful? I've heard that the best way to see the fragments is to apply them to an 1% agarose gel and run it at 80V for 4h, do you agree?

Thanks in advance!


M Jones

#2 perneseblue

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Posted 06 January 2011 - 09:32 PM

When digesting DNA fragments for cloning, I usually run the digest overnight. Thus regardless of the amount of DNA, the digest will have sufficient time to go to completion. But please note, while most common restriction enzymes are active for prolong periods, some of the more exotic enzymes are unsuitable for long digest.

As to how much DNA to digest... I am very wasteful with DNA. I believe in the idea of cut once and never do it again. DNA is there to be use, so be generous. I tends to cut enough DNA such the fragment of interest is approximately 5ug. Qiagen gel purification kit has a maximum binding affinity of 10ug. Be generous. It is better to have too much then too little. And qiagens can be decontaminated with HCl, re equilibriated and reused.

As for how much enzyme to use...
there is one rule of the thumb, the total volume of restriction enzyme used should not be more than 5% of the total digest volume.
eg: A 100ul digest should only have a total of 5ul of restriction enzyme

This is due to glycerol. Glycerol is used a preservative for the enzyme. Unfortunately, glycerol also inhibits the enzymes activity. Too much glycerol in the digest, the enzyme won't work or worse start exhibiting star activity. Ie it start cutting at site similar but not identical to its DNA recognition sequence.

The NEB website's technical guide is also very useful for information of the cutting efficiency of a number of RE .



The running conditions of the agarose gel depends on size of the DNA fragment of interest
Below is what I use.
150-300bp ~ 2.5% TAE agarose gel, run at the highest voltage available
300-900 bp ~2% TAE agarose gel, run at the highest voltage available
1kb-3kb ~1% TAE agarose gel run at 90-110V
3kb-5kb ~0.7% TAE agarose gel run 60-70V

The slower (lower voltage setting) you run the gel, the better the separation of DNA.
But the slower you run the gel, the longer it takes (bad for you) and the longer it take the more the DNA can diffuse laterally, making the bands less sharp.
The faster you run the gel, the hotter the buffer becomes.
The lower the percentage of agarose, the easier it is to melt.

All this means, to purify small bands, run gel fast and use high percentage agrose gel.
To resolve big DNA bands, run gel slow.and use low percentage agarose gel.
May your PCR products be long, your protocols short and your boss on holiday

#3 mjones

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Posted 07 January 2011 - 02:59 AM

Thanks for your comment and help.

But the thing is... I don't know the size of my fragment of interest because I don't know the content of the plasmid... that's what I'm trying to know... So I don't know if I want to resolve large or small fragments, mas I'm sure that the fragment will be larger than 1100bp because I want that the fragment include my unkown gene of interest....Do you understand? In these case how do you would proceed? I hope that by making the ligation of all fragments randomly to a PBK-CMV vector, making the electroporation and selecting the gene (the gene is responsible for antibiotic resistance so it's easy to select the fragment in which is included) I will find and know the gene.

And the there is another thing: I have always problems when I quantify my plasmidic DNA because I obtain one thing by visualization in agarose gel and another thing when I quantify the DNA by spectrophotometry. In your case how much volume of DNA do you add to the digestion reaction to obtain a 5ug concentration in a 100ul total reaction volume?

Thanks again!




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