Ligation

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Thu Jun 21 22:35:37 EST 2007


MICHAEL L SULLIVAN wrote:
> 
>> The most important thing is having good competent cells.  
>> Transformation 
>> efficiency of over 10^8 is ideal.  And no CIP.
>>
> 
> While it's important to have good competent cells, for routine cloning, an efficiency of >10^6/ug shoud be more than adequate. That's my expererience anyway.
> 
> Mike Sullivan
> 

10^6 rather low (I'd never use anything less than 5 x 10^7 for cloning), 
and I suspect it is more suitable for cloning in cloning vector (which I 
don't use), but I can understand why.  There are many variables in 
ligation (e.g. concentration and size of DNA, vector/insert ratio etc.), 
and this is the bit I'd confess -  I tend to be a bit slapdash in what I 
do and just ignore the variables.  For example, I'll just look at a gel 
and say "oh, 2 ul of that will do" and never really bothered to 
calculate the DNA concentration or even check if I have any DNA after 
purification (I only get the students to do it when teaching them).  So 
I'll get 2000 colonies from one ligation, but maybe 10 in another.

Having good competent cells just makes life easier - I can do just a 
single ligation reaction for each construct (easier when I am making 
half a dozen or more different constructs each time) but still be sure 
of getting most (if not all) of them done at the end of the week.  I've 
done a few hundred constructs this way, and I'd say having good 
competent cells is the main reason why I only need to do repeat ligation 
for a very small fraction of them.


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