competent cells transform 12 kb plasmid,urgent - (May/23/2007 )
I tried to transformed my 12kb plasmid into DH5 alpha cells,but did not get any colonies. Any suggestions of competent cells for transforming such large plasmid?
It should normally work in DH5 alpha. verify the antibiotic resistance and also may be the plasmid contains some toxic components which might require you to transform in some special cells.
You could try XL-gold cells from stratagene.
i have experience to transform 15 kb plasmid in DH5a. transformation frequency was as usual as shorter plasmids
Heath-shocked >15 kb into DH5a without any problem. (also worked with other competent cells without too much problems).
heat shock is better. I used also double electric choc for increasing transfert and worked well
Thank you very much for your reply. Acturely, i am trying to transform RCAN vector plus the gene of interest. Does anyone have any experience on the transformation of RCAN(12kb)?
Out of curiosity, what is double electric choc?I am unfamiliar with that technique. Thanks.
well it's a way to say that i'm electroporate bacterias. But instead of adding LB and transfert to 1.5ml tube for recovery and growth, i redo an electric choc. Then proceed as usual with rotation 300rpm 37° but for 1H
i was so desperate with that plasmid so i tried differents things without any scientific reference sometimes (and "double eletric choc" is one of tehm )
electroporation is always better than transformation. I tried with 10kb plasmid, no problem. Just make sure with the washing steps with 10% glycerol should be 3 times.