In article <32F05C68.4369 at sheffield.ac.uk>, K.Mulcahy at sheffield.ac.uk wrote:
>> I have just performed a blunt-end ligation for the first time using
> 160ng of a 1.635kb insert and 343ng of a CIAP-treated 10.5kb vector
> (therefore a 3:1 molar ratio of insert to vector). The total volume of
> the ligation mix was 40µl and contained 2 units of Gibco's T4 DNA
> ligase. The mixture was incubated at room temp for 24 hours.
>> Since I have not done such ligations before, I was wondering how much of
> the ligation mix to use to transform my bacteria (XL1-blue). In order to
> maximise the chance of obtaining colonies I am considering precipitating
> the ligation mix and redissolving it in a smaller volume and adding the
> whole lot to the transformation. Is this okay or would you advise
> against it?
>> Also, does anyone know the answer to the following question? When adding
> your ligation mix to the bacteria (without precipitation of the DNA
> first) why is it recommended (e.g. in the Gibco T4 DNA ligase protocol)
> that you dilute the ligation mix more than 5 times before adding it to
> the bacteria?
>> Many thanks,
>> Kevin Mulcahy.
There was a report in Biotechniques last month (?) which discussed using
an agarose well prepared in a microfuge tube to clean DNA before
ligation. It looked very simple to do and the authors reported (if I
remember correctly) using 40 ul of ligation to electroporate! Their
efficiency was incredible. Hope this might help. Also, the ligase (as
well as other enzymes) are stored in glycerol which will inhibit the
enzyme if not diluted.