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Competent Bacteria Preparation

Competent Bacteria Preparation - Protocols and Methods Forum

Competent Bacteria Preparation - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 06-02-2010, 08:03 PM
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Default Competent Bacteria Preparation



Hi there ^^

I want to share with you this protocol. It's quite effective, so you can prepare bacteria as competent as 5x10^6 cfu/ug DNA (it's the most common value I've got with this protocol), with little material, and get plenty of single use aliquots, so you can avoid freezing/thawing cycles which lower the efficiency.

Solutions required:

CaCl2 0.1M, ice cold and filtered
MgCl2 0.1M, ice cold and filtered
Storage solution: 85% CaCl2 0.1M, 15% Glycerol, ice cold and filtered

You'll be needing a centrifuge capable of 4000 rpm at 4C, and 50mL falcon adapters

Some advice before starting: Gather ice... plenty of ice, because it's a protocol that lasts 3-4 hours to complete, and the whole time you MUST have the bacteria and the solutions on ice

1.- Culture your bacteria overnight in 4mL of your growing medium. A 1:1000 bacteria:medium inoculation works great.

2.- Inoculate 200 mL of new, sterile growing medium with 2 mL of your overnight culture and grow it until OD600 ~ 0.6

*From now on, everything must be done on ice. Falcons and Eppendorfs must be ice cold when used*

3.- Put the 200mL of the culture in four 50mL falcon and place them on ice for 10 minutes.

4.- Centrifuge 10 minutes at 3000 rpm at 4C. Discard supernatant and place the pellet quickly on ice.

[From now on, every volume given for resuspension must be used for every 50mL of culture you had]

5.- Gently resuspend the pellet in 12.5 mL ice cold MgCl2 0.1M. The more glently you resuspend them, the greater the efficiency.

6.- Incubate on ice for 5 minutes

7.-Centrifuge 10 minutes at 4000 rpm at 4 C. Discard supernatant and place the pellet quicky on ice.

8.- Gently resuspend the pellet in 2.5 mL ice cold CaCl2 0.1M

9.- Incubate on ice for 20 minutes

10.- Centrifuge 10 minutes at 4000 rpm at 4C. Discard supernatant and place the pellet quicky on ice.

11.- Gently resuspend the pellet in 1 mL storage solution. Aliquote in 100 uL fractions in 1.5 mL ice cold eppendorfs, and store directly at -80 C.


Hope it helps!

Best Regards

Last edited by SebaQ; 06-03-2010 at 02:02 PM.
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