Day 1: Innoculate the cells in 10ml LB Medium and Grow Overnight.
Day 2: From the O/N culture Freshly innoculate 0.1 - 0.5% Innoculum into 100ml of LB Medium and start growing the cells freshly till the O.D of the Culture reaches 0.4 - 0.5 at A600nm.
1) Once the O.D was reached incubate the cultore at 4 degrees for 30 minutes
2) Take the culture into two 50ml centrifuge tubes and spin down the cells ( Maintain Centrifuge at 4 degrees) ( 5000rpm - 5mins)
3) Discard the sup and resuspend the cells in 1/2 the original Volume (25 ml) of ice Cold 0.1M MgCl2 and and incubate in ICE for 15 mins
4) Centrifuge the cells and discard the sup ( 5000rpm - 5mins)
5) Again resuspend the cells in 1/2 the original Volume (25 ml) of ice Cold 0.1M CaCl2 and and incubate in ICE for 15 mins
6) Centrifuge the cells and discard the sup ( 5000rpm - 5mins)
7) Resuspend the cells in 1ml of ice Cold 0.1M CaCl2+20% Glycerol and aliqoute 100ul into each 1.5 ml tubes and store them immediately in -70 degrees
8) One Vial you can check for competancy immediately by doing Transformation.
Day 1: Innoculate the cells in 10ml LB Medium and Grow Overnight.
Day 2: From the O/N culture Freshly innoculate 0.1 - 0.5% Innoculum into 100ml of LB Medium and start growing the cells freshly till the O.D of the Culture reaches 0.4 - 0.5 at A600nm.
1) Once the O.D was reached incubate the cultore at 4 degrees for 30 minutes
2) Take the culture into two 50ml centrifuge tubes and spin down the cells ( Maintain Centrifuge at 4 degrees) ( 5000rpm - 5mins)
3) Discard the sup and resuspend the cells in 1/2 the original Volume (25 ml) of ice Cold 0.1M MgCl2 and and incubate in ICE for 15 mins
4) Centrifuge the cells and discard the sup ( 5000rpm - 5mins)
5) Again resuspend the cells in 1/2 the original Volume (25 ml) of ice Cold 0.1M CaCl2 and and incubate in ICE for 15 mins
6) Centrifuge the cells and discard the sup ( 5000rpm - 5mins)
7) Resuspend the cells in 1ml of ice Cold 0.1M CaCl2+20% Glycerol and aliqoute 100ul into each 1.5 ml tubes and store them immediately in -70 degrees
8) One Vial you can check for competancy immediately by doing Transformation.
I would recommend the method described by Inoue, H. et al. (1990) Gene 96 23-28. We used the attached protocol, which works pretty well.
In the end, most protocols designed to do this work work pretty well. The absolute key is making sure there is no detergent in any flasks you use for the bugs. Your success will correlate to the degree to which you solve that issue. If you have any glassware that has never been used, try that next to your washed glassware. I would like to place a side bet on the new glassware.
Hi Kapoor
I have attached here a protocol I regularly use and I never failed to make competent cells. I think you can follow that.
Best of luck
Rezaul
I would suggest the attached protocol for heat shock method.
All the best
Most protocols will work fine, the absolute key for super competence is to use glassware with no detergent residue. New glassware out of the box will give you a log increase over extremely well washed glassware, which will significantly outperform glassware in which you didnt take care to rinse any residual detergent from the containers.
Hi, I follow a similar protocol than those described above. Rather than Cold 0.1M MgCl2 I use 15 ml and wash with a sterilized 10 % glycerol solution. At the final step, the cell pellet is suspended in 40 ul of 10% glycerol and transferred to an Eppendord. The cells can be immediately used for electroporation (add 1 ul of your plasmid/linear DNA on ice and transfer to cold electroporation cuvette) or be stored at -80°C.
Comment: Please cite centrifugation steps in xG rather than RPM. Makes a huge difference even in the same machine using different rotors (eg swing out rotor 2500Xg versus fixed vs 1600xg!
Hi, I would like to know if anyone has tested the protocol for competent cells of "Kinnera Naraganeni" and know the efficiency. Thank you
Here is the protocol suggested by Addgene:
https://blog.addgene.org/save-time-and-money-by-making-your-own-competent-cells
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