E. coli steps:
(1) Adjust the temperature of the constant temperature water bath to 42 ° C in advance.
(2) Take a tube (100μl) of competent bacteria from the -70 ℃ ultra-low temperature freezer, immediately heat it with your fingers to melt it, insert it on the ice, and place it on the ice bath for 5 ~ 10min.
(3) Add 5μl of the connected plasmid mixture (DNA content does not exceed 100ng), shake gently and place on ice for 20min.
(4) After gently shaking, insert into a 42 ° C water bath for 1 ~ 2min for heat shock, then quickly put it back into the ice and let stand for 3 ~ 5min.
(5) Add 500μl of LB medium (without antibiotics) to the above tubes in the ultra-clean workbench and mix gently, then fix to the spring rack of the shaker and shake at 37 ℃ for 1h.
(6) Take 100 ~ 300μl of the above transformation mixture in an ultra-clean workbench, and drop them into a solid LB flat Petri dish containing appropriate antibiotics, and apply evenly with a glass coating rod burned with an alcohol lamp (note: glass coating After the alcohol on the stick is extinguished, wait a while, and then apply it after it has cooled down).
(7) If the carrier and host bacteria are suitable for blue and white spot screening, add 40μl 2% X-gal and 8μl 20% IPTG on the plate after the bacterial solution is dripped, and apply evenly to the glass coating rod burned with alcohol .
(8) Mark the coated petri dish and place it in a 37 ° C constant temperature incubator for 30-60min until all the liquid on the surface penetrates into the medium, then put it upside down and put it in a 37 ° C constant temperature incubator overnight.
(9) Spray 70% ethanol on the desktop contaminated with bacteria, dry the desktop, and write an experiment report.
(10) Observe the colony colonies growing on the plate, it is better to separate the colonies from each other. Note the white plaque.
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