drm21 at mole.bio.cam.ac.uk
Thu Jul 6 13:52:58 EST 1995
In article <950706.105428.18791 at macpost.lidac.liu.se>, SaiSu at MCB.LiU.SE
(Sailesh Surapureddi) wrote:
> >From: tkuwada at neuron.uchc.edu (TimHusky)
> >Iam having trouble with a seemingly straight forward sticky-end
> >ligation. I have
> >used up to 800U/20ul rxn with Insert:plasmid ratio's of 3:1 - 13:1,
> >total DNA of
> >up to 300ng. These rxn have been overnight @ 16C.I have several
> >1) Am I using too much or too little DNA?
> : No, you are not using too much, but why waste insert and
plasmid, reduce it
> two 2:1 ratio, besure to denature them both before adding the ligase
Denature them? What on earth for? I never do this.
> >4) Will spiking my buffer w/extra ATP help & what is the deal w/ PEG,
> >does it increase ligation efficiency
> :No, again, it does not increase the ligation efficiency. But you can
> ligation mix with Et-OH for added safety for transformations.
Again, I'd say this was totally unnecessary unless he is doing
electroporations, where the salt in ligation mix may cause problems.
Also, the ATP can go off in very old/badly kept ligation buffer, so adding
fresh _may_ help.
> >5) How can I increase the competency of my DH5-alpha cells,Iam
> >currently using a 50mM CaCl2 2X wash of cells at .3-.4OD @ 590nm
> :The ligations conditions have not been described by you, you could try this
> (incubating the competent cells with insert and vector on ice for 30min
> shock at 42 for 5 min and again on ice for 5 min). That should do the trick.
> >I would greatly appreciate any tips.
42 for 5minutes?!!! 42 for 45 seconds is far more usual (in Falcon 2059
tubes). Personally, I usually use 5 minutes at 37C in eppendorf tubes,
since it is more convenient here and works well. Not so different I
suppose. As I suggested to Tim by Email, making better competent cells
is probably worth the effort - the Inoue et al method (Gene 96,
23-28,1990) is good, and has been recommended here several times.
> One more: the ligation could increased and the time decreased by
> ligation mixture in sonication water bath for 15min at 16 C, this works very
> well in my case and now I get lot of colonies.
I remain deeply sceptical about this, (and microwave restriction
digests). Has anyone on the net actually done a side-by-side comparison
of sonicated vs unsonicated ligations? If so, please post the results!
FWIW I usually only ligate for 15 minutes at room temperature for
sticky-ended ligations; 1 hr for blunts. They usually work fine anyway,
without sonication. (I use NEB ligase and buffer. About 0.1ul ligase/10ul
reaction. No connection with NEB, but their ligase is great!)
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