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Hello,
Trả lời:
maud63 wrote:I would like to know how you make your bacterial transformation :
- electroporation or chemically competent cells?
- Do you purify your ligation reaction before use (and how?) or do you directly use it? Which maximum volume of ligation reaction for which bacterial volume?
- how many ng of DNA?
- Do you thing it is better to heat inactivate the ligase before transformation?
Thank you for your tips to have the best transformation efficiency.
Trả lời:
scolix wrote:If u have an apparatus for electroporation then go for it, as it gives better transformation.
I donot purify my ligation mixture but use 2ul-3ul (out of a total 20ul) of the ligation mixture for transforming chemically competent cells. If its electroporation then 1-1.5ul.
The amount of DNA varies a lot in my liagtion. Normally I use between 20-40ng in my ligation but I have used upto 200ng for ligations.
I donot think it as necessary to inactivate ligase before transformation. As I leave the ligation mixture on the bench so that I could use it the next day if something was went wrong.
-scolix-
Jou wrote:I remember cloning with TOPO vector from Invitrogen using electro-competent cells. Didn't work, because I didnt' purify the ligation and it still contained too much salt from the Invitrogen-supplied ligation buffer.... but usually I don't purify.
-Jou-
phage434 wrote:Our tests show that chemical transformation with ligation mixes should add less than 5% by volume of ligation mix -- more is inhibitory. We routinely use 1 ul with 20 ul of cells. Heat killing ligase appears to be optional from our experience, but fatal if you are using PEG containing ligation buffer (quick ligation kits). We do not purify our ligation reactions, although we could probably get more transformants by doing so. For electroporation, the main limitation on added ligation mix is the increased conductivity of the cells, which can lead to arcing. Dialysis on floating Millipore membranes over TE or water helps a great deal in this situation.
Concentration of the DNA in a ligation reaction is a major factor in pushing the reaction from intra-molecular to inter-molecular. We usually want low concentrations, since we want to avoid formation of concatamers, which form easily, do not transform well, and are not the desired product. The optimal ratio is a 1:1 molar ratio of insert:vector. We aim for around 20 ng of vector in the 10 ul ligation mix.
-phage434-
fred_33 wrote:i use 1/10 of my ligation reaction (for up to 20µl) directly for electroporation.
For heat shock cells, i use the whole ligation directly.
For the TOPO cloning, i use 1 to 2µl of a 1/10° diluted topo reaction for electrocompetent coli.
Finaly, i precipitate ligations with butanol.
-fred_33-
