1. Protocol of Competent cells formation (BL-21)
SARFRAZ-UR-RAHMAN
PH.D Scholar (Parasitology)
References:
1- Cloning and DNA analysis 6th Edition by TA Brown
2- Preparation of calcium competent Escherichia coli and heat-shock transformation
(JEMI methods) Chang, et al. 2017
3-Internet source
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2. Competent cells
• A cell ̓s ability to take up foreign or extracellular DNA from its surrounding environment.
The process of genetic uptake is called transformation
• Difference between competency and transformation.
• Competency.. A cell ̓s ability to take up foreign or extracellular DNA from its surrounding
environment
• Transformation……The action of competent cell to taking up the genetic material.
• Types…
• Electro-competent cell
• Chemical or CaCl2 or Hanahan ̓s method competent cells
3. Procedure for Electro-competent E.coli Cells
BL-21
Pick single
colony
10 ml LB-S
100 ml LB-S
50 ml Falcon tubes
At shaker incubator, at
37 ˚C , 220 rpm
Overnight
At shaker incubator, at
37 ˚C , 22O rpm for 3-4 hrs., OD=0.3-0.5
BL-21 cells on
LB-S agar
plate
1 ml culture
save at 4 °C
4. Procedure of Electro-competent E.coli Cells
Centrifuge at 7000 rpm at 4 ˚C for 5 min
Discard the supernatant
Resuspend the pellet in 1ml of sterile, ice-cold dd H2O
Transfer it into 1.5 ml microcentrifuge tube
Total repeat these steps 3 times
4th time----Centrifuge at 7000 rpm at 4 ˚C for 5 min Discard the supernatant
Add the 50 µl of sterile,
ice-cold dd H2O
Substitute 10 % glycerol for
sterile, ice-cold dd H2O for long
term storage at or -80 ˚C or above
Use ice for tubes and
work under flame
during the protocol
5. Procedure of Chemical or CaCl2 or Hanahan ̛s
method E.coli Cells
• Step I:
• Whole environment…make sterile
• Cleaning the flame area
• Do alcohol spray
• After 10 min of flames….pouring will be started
BL-21
Pick single
colony
BL-21 cells on
LB-S agar
plate
2 ml LB-S
broth in 15 ml
falcon tube
At shaker incubator, at
37 ˚C , 220 rpm
Overnight
Flame
6. Procedure of Chemical or CaCl2 or Hanahan ̛s
method E.coli Cells
• Step II: Subculturing
• Work under the flame conditions
Flame
1 ml culture
save at 4 °C
At shaker incubator, at
37 ˚C , 22O rpm for 3-4 hrs, OD = 0.4
99 ml LB-S (1:100)
7. Procedure of Chemical or CaCl2 or Hanahan ̛s
method E.coli Cells
Flame
Put the tube into ice
box for 20 min
Ice box
Secondary culture of
BL-21
Pour 20 ml into 50 ml
falcon tubes
Step III: use ice for tube and work
under flame area
8. Procedure of Chemical or CaCl2 or Hanahan ̛s
method E.coli Cells
Centrifuge at 4000 rpm at 4 ˚C for 10 min
Discard the supernatant
Resuspend the pellet into 20 ml of sterile, ice-cold 0.1 M
CaCl2
Incubate it into ice for 30 min
Convert it into the 50 µl
volume in each 2 ml
cryopreserve vials
Use ice for tubes and
work under flame
during the protocol
Centrifuge at 4000 rpm at 4 ˚C for 10 min
Indication:
Pellet will be
more diffused
than previously
(My
biosource.com)
.
Discard the supernatant
Resuspend the pellet
into 5 ml of sterile,
ice-cold 0.1 M CaCl2
with 15 % glycerol
Store 20 cryopreserve
vials of competent cells
into Liquid Nitrogen
container
