IGEM:IMPERIAL/2009/XL-1 Blue QC

Quality Control:

Quality Control Rational:

XL-1 Blue E.coli must satisfy two criteria:

1) They must be competent

2) The stock must not contain any antibiotic resistance.

To investigate competence, cells were transformed (via electroporation) with 1ng of DNA (plasmid conferring kanamycin resistance).

Protocol:

1) Add 10µl of kanamycin plasmid to 50µl of stock XL-1 Blue culture and pipette up and down to mix.

2) Transfer 50µl of DNA + E.coli mix to an electroporation cuvette.

3) Add 450µl of LB and pipette up and down.

4) Electroporate.

5) Remove 450µl from electroporation cuvette and transfer to a sterile Eppindorf tube.

6) Spread 5µl and 50µl of the transformed cells onto two different agar plates containing kanamycin.

7) Incubate overnight.

To investigate the presence of any unwanted antibiotic resistance the cells were plated onto antibiotic containing agar plates.

Protocol:

8) Transfer 50µl of stock XL-1 Blue culture to an electroporation cuvette.

9) Add 450µl of LB and pipette up and down.

10) Electroporate.

11) Remove 450µl from electroporation cuvette and transfer to a sterile Eppindorf tube.

12) Spread two antibiotic plates (one kanamycin & one ampicillin) each with 50µl of cells.

13) Incubate overnight.