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topo ta cloning

topo ta cloning - Protocols and Methods Forum

topo ta cloning - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 12-26-2004, 09:46 PM
as
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Default topo ta cloning



Hi. I have a few basic questions regarding topo TA cloning

1) When I TA clone a taq produced PCR fragment into a given TA topo vector
and subsequently sequence or colony screen the clones I usually find that
most of the clones are in the same direction? But there should be random
orientation of the fragments resulting in a 50/50 distribution?

2) After heatshocking the oneshot cells + ligation mix and adding SOC, how
long time (minimum) does the mix need to shake at 37 degrees? As it is AMP
selection I guess only around 10 min are needed, however the manual says one
hour? Could I go down to 10 min, and how will this affect the cloning
efficiency?
Thanks
AS
DK


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  #2  
Old 12-27-2004, 07:24 AM
D.K.
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Default topo ta cloning

On Sun, 26 Dec 2004 22:46:55 +0100, "as" <[Only registered users see links. ]> wrote:


I don't have an explanation but I've seen the same. Seesm to be insert-specific?
In some cases, over 90% are in one irionetation, in others - as expected,
roughly 50/50.


If it's Amp then no recovery is necessary! It WILL happen on dishes. You
migth recall that Amp does not kill cells - only prevents them from dividing.
So, they will sit still on dishes for ~ one hour to synthesize enough
lactamase and then will go on to divide. I've done tests back to back
and the difference, where it exists, is marginal and can 100% be accounted
for by the cell division without antibiotic during the recovery time.

Of course, with kanamycin and the other stuff that poisons cells,
the recovery is a must.

DK

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  #3  
Old 12-27-2004, 12:38 PM
Jose de las Heras
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Default topo ta cloning


"D.K." <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...

I once added ampicillin by mistake to the recovery medium, for an hour, then
plated... and I got exactly zero colonies. Of course it could have been a
bad transformation... but I doubted it as I had been doing transformations
almost daily with the same cells without a hitch. I blamed the ampicillin,
so I find your results interesting: you really tried back-to-back (ie: amp
vs. no amp post transformation) and had good growth even with amp right
after transformation? I wonder what went wrong in mine...

Jose

Jose


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  #4  
Old 12-29-2004, 08:17 PM
ChenHA
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Default topo ta cloning



"D.K." wrote:

The reason is usually toxicity of the gene product.



Ampicillin is bactericidal - it causes cell lysis when the cell tries
to expand and divide. So it is only bacteristatic when the cell is at
stationary phase. Some may survive as spheroplasts or filaments
(especially if the concentration of the antibiotic is not high), but
they are unstable.

However you are right in saying the recovery step is unnecessary, I
normally don't bother. The cell wall is constantly being broken down
and rebuild in E. coli when it is growing, but the effect of the
antibiotic takes time to completely destabilise the cell wall and
cause cell lysis, and in that time, cells containing the
beta-lactamase gene would have time to synthesise the protein to
counteract the antibiotic.



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  #5  
Old 12-30-2004, 02:42 AM
D.K.
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Default topo ta cloning

On Wed, 29 Dec 2004 20:17:09 +0000, ChenHA <[Only registered users see links. ]> wrote:


I imagine that could be the case but it's not the explanation I like. The
plasmids we are talking about are straight cloning vectors with no promoters,
ribosome binding site, etc. The toxicity would have to be extreme - which is not
the case of the same genes expressed later in pETs.

DK

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  #6  
Old 12-30-2004, 09:23 AM
as
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Default topo ta cloning

Hi again.
Thanks for the answers.
I decided to test the difference between recovery and no recovery.
So I transfected 50 Ál of invitrogen top10 cells with 1Ál of ligation mix,
and after heatshock I plated 25Ál on amp plates and the other 25Ál I
incubated with SOC at 37 for 40 min.
On the first plate I got around 30 colonies and on the other plate, around
300. So I guess it is worth doing after all!
Happy New year.
AS

"as" <[Only registered users see links. ]> wrote in message
news:njGzd.640$[Only registered users see links. ]...
one


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  #7  
Old 12-30-2004, 07:27 PM
ChenHA
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Default topo ta cloning



"D.K." wrote:

I haven't tried Topo TA cloning before (in fact, I always clone
directly into expression vector, and none of these expensive Topo
stuff), so I can't say what what might be the cause exactly in this
case. I have found that, however, vectors that aren't supposed to
express a protein often do, albeit in tiny amount. The cells may only
be growing somewhat slower, i.e. there might be the same number of
clones in both orientation but the colonies are much smaller or not
visible the next morning.



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  #8  
Old 12-30-2004, 07:44 PM
ChenHA
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Default topo ta cloning



as wrote:

The difference might be smaller than you think. At an optimum growth
rate of around 20 minutes doubling time, only a quarter of the 300
colonies you see may actualy be transformants. Personally I found the
differences to be minor, and normally unimportant unless the ligation
you do is difficult.


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  #9  
Old 12-31-2004, 12:04 AM
D.K.
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Default topo ta cloning

On Thu, 30 Dec 2004 19:44:30 +0000, ChenHA <[Only registered users see links. ]> wrote:


As I know the difference in my case was no more than 2X, I could also
think that there might be a difference between chemical transformation
(as in above) and electroporation (that I use). If "chemical" cells are
"sicker" then perhaps they are more sensitive to the weak bactericidal
activity of Amp?

DK

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  #10  
Old 12-31-2004, 07:06 PM
ChenHA
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Default topo ta cloning



"D.K." wrote:

I can't really say, since I use only chemical methods, but I shouldn't
think blasting holes in the cells in electroporation would do the
cells much good either. But I do think that some cell strains are
probably worse affected than others, for example, I suspect XL1 Blue
has weaker cell wall already and may do worse in this respect, but I
never had much problem DH5alpha. If incubation is necessary, I think
10 minutes is probably all that it needs for ampicillin selection.


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