Ligation, "quick" vs plain/normal
Nathan McCorkle
normal ligase? It seems like its in the buffer, so what is it then
that quickens things? Why would one want to avoid a quick ligation?
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Cory Tobin
> normal ligase? It seems like its in the buffer, so what is it then
> that quickens things? Why would one want to avoid a quick ligation?
The difference is the buffer. The quick ligase buffers have PEG 6000
(or some other large PEG polymer). PEG increases the rate at which
the reaction works. The downside is that you can't heat kill the
ligase because of the PEG. Apparently heating up PEG completely
inhibits the transformation.
A while back it was shown that heat killing the ligase increased the
transformation efficiency, at least in electroporated cells. To my
knowledge no one ever studied the effect of heat killed ligase on
chemical transformation.
My suggestion is to use the quick ligase with no heat kill step. But
if you are having very low transformation rates switch to the regular
T4 ligase buffer with no PEG 6000, do the ligation for a longer period
of time, heat kill the ligase, then transform.
-Cory
Nathan McCorkle
messes with downstream restriction digests if the PEG is heated? For
an upcoming experiment I'll likely be doing gel elutions, so I'll have
columns on hand to rinse off any PEG... ethanol precip would probably
do the trick too, right?
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General Oya
What might be a good ligase to turn into a part? We have been considering doing TAq since we could still use a boiling purification (I think)... But I didn't know what other considerations might be at work? Microbial versus Yeast?
Ryan
General Oya
Nathan McCorkle
http://www.neb.com/nebecomm/products/productM0202.asp
http://www.neb.com/nebecomm/products/productM0208.asp
T4 has a lot more functionality.
On Wed, Mar 30, 2011 at 12:48 AM, General Oya <gener...@gmail.com> wrote:
Cory Tobin
> messes with downstream restriction digests if the PEG is heated? For
> an upcoming experiment I'll likely be doing gel elutions, so I'll have
> columns on hand to rinse off any PEG... ethanol precip would probably
> do the trick too, right?
In my experience using PEG works better. Another thing that I have
found to increase the ligation/transformation efficiency is to use 2x
ligation buffer instead of 10x.
Not sure about the digestion. I've never done a ligation followed by a
digestion. But I imagine if you column purify or precipitate, that
would work fine.
-Cory
Cory Tobin
T4 is the most common. Biobricks don't use blunt ends so you could
probably get away with using E. coli DNA ligase instead, but everyone
has T4 in their freezer.
> What might be a good ligase to turn into a part? We have been considering
> doing TAq since we could still use a boiling purification (I think)... But I
> didn't know what other considerations might be at work? Microbial versus
> Yeast?
If you're just going to use it to ligate sticky ends, T4 is your best
bet. there are other ligases out there but they are for specialized
situations.
-Cory
Nathan McCorkle
>> have you experience both with and without PEG? Do you know if it
>> messes with downstream restriction digests if the PEG is heated? For
>> an upcoming experiment I'll likely be doing gel elutions, so I'll have
>> columns on hand to rinse off any PEG... ethanol precip would probably
>> do the trick too, right?
>
> In my experience using PEG works better. Another thing that I have
> found to increase the ligation/transformation efficiency is to use 2x
> ligation buffer instead of 10x.
>
2X because pipetting error affects the end concentration less?
> Not sure about the digestion. I've never done a ligation followed by a
> digestion. But I imagine if you column purify or precipitate, that
> would work fine.
>
>
> -Cory
>
Jordan Miller
jordan
Nathan McCorkle
> fyi: ethanol precipitation to get rid of the PEG probably won't work. some or most of the PEG will precipitate in ethanol too (depending on MW, temp, freezing steps, and how much water you have left).
Will PEG migrate to an organic layer? i.e. Chloroform??
Emmette Hutchison
Adding a bit of fresh ATP to the mix also improves ligation efficiency (this isn't strictly necessary if you ), as does PNKing if you are trying to ligate PCR products.
Emmette
-Cory
Nathan McCorkle
> I'll second the recommendation on PEG improving ligation efficiency. Heating
> inactivating does cause some bad mojo in terms of transformation. Not sure
> if EthOH precipitation or silica beads will remove it.
>
> Adding a bit of fresh ATP to the mix also improves ligation efficiency (this
> isn't strictly necessary if you ), as does PNKing if you are trying to
> ligate PCR products.
FYI, PNK = polynucleotide kinase... swaps a 5' OH to be a 5' phosphor
--
Cathal Garvey
I'm with you on the buffer: 10x buffer for ligase is a precipitation nightmare, often requires loads of vortexing to resuspend/redissolve contents of the buffer. Failure to fully suspend stuff at least once will permanently change the constitution of the buffer for all later uses, so I'd suggest going for a lower concentration buffer and avoiding the problem entirely.
We were given a workshop on cloning tips by the good folk of fermentas, and apparently regular ligase gets most of the work done after only 10mins at room temperature, so that's already pretty 'quick' for me. Heat inactivation of Ligase does increase cloning efficiency, but no mention of PEG was made in that talk.
Anyone here know why PEG increases efficiency? It's used in transformations of Yeast etc. apparently because it puts mild osmotic shock on the cells, I suppose that means it's hydrophilic and creates a large shell of hydration? Does this effectively concentrate the remaining free water or something, or act as an osmotic "buffer" to concentrate things without going so far as to induce precipitation?
Does the MW of the PEG used have any significance, besides the amount you may have to add to achieve the same effect? Because some high MW PEGs may freeze-out of water at lower temperatures, while other low MW PEGs may have enhanced solubility in alternative solvents, I imagine either way there are different means of purifying the DNA out of the stuff.
You could always gel purify the DNA if you felt patient; PEG isn't charged much if at all, so it shouldn't migrate with the DNA.
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On 30 Mar 2011 06:26, "Cory Tobin" <cory....@gmail.com> wrote:
> have you experience both with and without PEG? Do you know if it
> messes with downstream restrict...
In my experience using PEG works better. Another thing that I have
found to increase the ligation/transformation efficiency is to use 2x
ligation buffer instead of 10x.
Not sure about the digestion. I've never done a ligation followed by a
digestion. But I imagine if you column purify or precipitate, that
would work fine.
-Cory
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You received this message because you are subscribed to the Google Groups "DIYbio" grou...
Jordan Miller
electrophoresis and gel extraction will get rid of all the PEG. PEG is completely neutral in charge, so it won't run during electrophoresis.
it probably won't matter to have some of it around though...
jordan
Nathan McCorkle
brush up on alcohol precip mechanisms!
Wouldn't the charge of PEG depend on pH? if it was high pH i would
think there'd be a negative charge, at least a little.
Jordan Miller
so PEG is really a great molecule!
i just checked google, DNA precipitates in ether:
http://www.transnetyx.com/Media/Files/DNA%20Isolation.pdf
jordan