Competent cells protocol using Zymo kit
(https://files.zymoresearch.com/protocols/_t3001_t3002_mix_go_e._coli_transformation_kit_buffer_set.pdf) :
Important! Each step of the following procedure should be done on ice or at 0-4°C.
1) Make 4 mL overnight culture (from a glycerol stock, or by picking a colony on a plate with the
corresponding antibiotic where the bacteria is spread and make a glycerol stock if there are
no glycerol stock)
When glycerol stock available:
-
Use a pipette tip to scratch the glycerol stock then put in 4 mL sterile LB medium. Shake
culture vigorously (usually 220 rpm) at 37°C (overnight).
When no glycerol stock available:
-
Spread 25 – 50 uL of competent cell aliquot onto an agar plate (without antibiotics), grow
at 37 °C overnight.
Pick one colony and put in 4 mL sterile LB medium. Shake culture vigorously (usually 220
rpm) at 37°C (overnight).
2) Use 0.5 ml of fresh, overnight E. coli culture grown in sterile LB (without antibiotic or with
the correct antibiotic if the bacteria is resistant) to inoculate 50 ml ZymoBroth™ or sterile
SOB medium in a 500 ml culture flask. Shake culture vigorously (usually 220 rpm) until the
OD600nm is 0.4 - 0.6 at 18°C (overnight).
tips:
- Zymobroth medium is limited, remember to keep around 0.5 ml for measuring the blank when
you monitor the OD,
- If the growing is too slow the day after, you can increase the temperature to maximum 26°C
3) Transfer the culture from Step 1 to ice. After 10 minutes, pellet the cells by centrifugation at
3,300 rpm (i.e., 1500 x g) for 10 minutes at 0-4°C.
4) Remove the supernatant and resuspend the cells gently in 5 ml icecold 1X Wash Buffer. Repellet the cells as in Step 2.
tips:
- The wash buffer and the competent buffers are given as 2x. Dilute them in the dilution buffer to
make 1x before spinning down the bacteria of step 2 to make sure that they are ice cold.
5) Completely remove the supernatant and gently resuspend the cells in 5 ml ice-cold 1X
Competent Buffer.
6) Aliquot (on ice) 0.05 ml of the cell suspension into sterile tubes. Cells are now ready for
transformation with DNA or can be stored below -80°C for transformation at a later time.
Remember to register the amount of fresh aliquots in GIN.
tips:
- After aliquoting, put the tubes in dry ice as fast as possible to freeze them.