questions about mung bean nuclease and ligation - (Mar/24/2008 )
To test if there is a hairpin in a plasmid, I need to digest the
plasmid with the mung bean nuclease.
Basically I am going to dilute the plasmid with TE buffer (10mM Tris
and 1mM EDTA), and then incubate the plasmid with Mung bean nuclease,
and at last stop the reaction by adding stop solution ( 3M ammonium
acetate and 250 u/ml tRNA).
My questions are :
What do EDTA, acetate ,and tRNA do, respectively?
I have never run a ligation product on the agarose gel .
What does it look like?
I mean the position of the ligation plasmid should be at the position
of a nick plasmid but not a supercoiled plasmid. Right?
And only s small amount of molecules are ligated. Right? So on gel it
is normal that we can not see ligation products. Right?
Mung Bean Nuclease requires some zinc for activty, so do not use too much EDTA. 1x TE will be OK.
Your "stop solution" is required for ethanol precipitation. Ammonium acetate gets DNA cleaner than sodium acetate because it is more soluble in ethanol.
tRNA is a carrier to aid in precipitation, but will interfere with subsequent 32P labeling. Use linear polyacrylamide for a completely neutral carrier.
Once Mung Bean Nuclease has nibbled the hairpin, you might need to repair the ends before ligation.
In 25 years of cloning, I have never bothered to run part of the ligation reaction on a gel. Transformation is a better assay.
You might also try T7 Endonuclease I (from New England Biolabs)
Nucleic Acids Res. 1990 October 11; 18(19): 5633–5636. PMCID: PMC332293
T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein.
B Müller, C Jones, and S C West
T7 endonuclease I is known to bind and cleave four-way junctions in DNA. Since these junctions serve as analogues of Holliday junctions that arise during genetic recombination, we have investigated the action of T7 endonuclease I on recombination intermediates containing Holliday junctions. We find that addition of T7 endonuclease I to strand exchange reactions catalysed by RecA protein of Escherichia coli leads to the formation of duplex products that correspond to 'patch' and 'splice' type recombinants. Resolution of the recombination intermediates occurs by the introduction of nicks at the site of the Holliday junction. The recombinant molecules contain 5'-phosphate and 3'-hydroxyl termini which may be ligated to restore the integrity of the DNA.
Other papers listed by Google search for "T7 Holliday"