Tuesday, July 8, 2008
To Johan
Hi Johan, thanks for reading my entry.
The method we use to transform the cells is: Heat Shock at 42 degree Celcius
This is how we do it:
1) Use 50-100µl of competent cells (E.coli), and add 0.5- 1µl of recombinant vector and incubate tube on ice for ½ hour. (keep the cells cold so that transformation efficiency will remain high).
2) Warm up agar plate that has amphicillin antibiotic, in a 37 degree Celcius incubator
3) Ensure water bath is warm up to 42 degree Celcius, ready for the heat shock process. (heat shock the cells so they open up their pores and DNA enters)
4) After ½ hour incubation, heat shock the mixture in step 1 for 45s in 42 degree celcius water bath, and immediately transfer it onto ice for 1 to 2 mins. (cool it immediately so that the pores closes back).
5) Add S.O.C medium (something similar to LB broth) in a 1:9 ratio. E.g. 50µl cells: 450µl of S.O.C .
6) After adding the S.O.C medium, put into 37 degree celcius incubator with shaker for 1 hour.
7) Obtain the transformed cells form the incubator, do a 50x dilution and plate it on the amphicilin plate.
8) Do 2 spread plate, one with the 50x dilution and an undiluted spread plate.
9) Allow both plates to stand for 1-2 mins then invert the plate and incubate it at 37 degree celcius for 1 day.
10) The following day, observe for any colonies formed. These cells are resistant to amphicilin, because of the amphicilin gene in the vector, acquired through the transformation process.
Blue White selection strategy (use to determine the presence of insert)
The blue white selection strategy makes use of alpha-complementation, which means the alpha and the beta part of beta-galactosidase is “completed”.
The diagram (above above) (duno why it appear at the top) is a lac-operon: lac operon is only switched on when glucose is absent and lactose is present.
IPTG (analog of lactose) is a gratuitous inducer of the lac operon.
Grow the colonies in the presence of X-gal, which is a colourless compound that can be cleaved by ß- galactosidase to produce blue colouration.
Hi Johan, thanks for reading my entry.
The method we use to transform the cells is: Heat Shock at 42 degree Celcius
This is how we do it:
1) Use 50-100µl of competent cells (E.coli), and add 0.5- 1µl of recombinant vector and incubate tube on ice for ½ hour. (keep the cells cold so that transformation efficiency will remain high).
2) Warm up agar plate that has amphicillin antibiotic, in a 37 degree Celcius incubator
3) Ensure water bath is warm up to 42 degree Celcius, ready for the heat shock process. (heat shock the cells so they open up their pores and DNA enters)
4) After ½ hour incubation, heat shock the mixture in step 1 for 45s in 42 degree celcius water bath, and immediately transfer it onto ice for 1 to 2 mins. (cool it immediately so that the pores closes back).
5) Add S.O.C medium (something similar to LB broth) in a 1:9 ratio. E.g. 50µl cells: 450µl of S.O.C .
6) After adding the S.O.C medium, put into 37 degree celcius incubator with shaker for 1 hour.
7) Obtain the transformed cells form the incubator, do a 50x dilution and plate it on the amphicilin plate.
8) Do 2 spread plate, one with the 50x dilution and an undiluted spread plate.
9) Allow both plates to stand for 1-2 mins then invert the plate and incubate it at 37 degree celcius for 1 day.
10) The following day, observe for any colonies formed. These cells are resistant to amphicilin, because of the amphicilin gene in the vector, acquired through the transformation process.
Blue White selection strategy (use to determine the presence of insert)
The blue white selection strategy makes use of alpha-complementation, which means the alpha and the beta part of beta-galactosidase is “completed”.
The diagram (above above) (duno why it appear at the top) is a lac-operon: lac operon is only switched on when glucose is absent and lactose is present.
IPTG (analog of lactose) is a gratuitous inducer of the lac operon.
Grow the colonies in the presence of X-gal, which is a colourless compound that can be cleaved by ß- galactosidase to produce blue colouration.
The alpha and the omega peptide must be “completed” for ß- galactosidase to be functional to cleave X-gal.
The lac Z alpha gene is found in the vector, and only when the vector is introduced into the lac Z alpha mutant (no alpha peptide) during the process of transformation, then there will be alpha complementation and produce functional ß- galactosidase and cleave X-gal to produce blue coloration. (Blue Colonies).
However, if there is an insert in the vector, because of successful ligation, the presence of the insert will cause disruption to the lac Z alpha gene and there will not be alpha complemention > no functional ß- galactosidase> no cleaving of X-gal >colonies remain white.
Jean Leong
TG02
=)
7:29 PM