Team:Amsterdam/Notebook/Protocols/Making Competent Cells

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Revision as of 12:29, 19 September 2011

Preparation of chemically competent cells

Overview

We use a slightly modified version of the chemical transformation protocol used by the [http://partsregistry.org/Help:Protocols/Competent_Cells Registry of Standard Biological Parts].
This protocol is a variant of the [http://www.ncbi.nlm.nih.gov/pubmed/1943786?dopt=Abstract Hanahan protocol] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [http://openwetware.org/images/b/bd/Pat6855494.pdf Bloom05 patent] as well. This protocol has been tested on NEB10, TOP10, MachI and [http://openwetware.org/wiki/Talk:TOP10_chemically_competent_cells BL21(DE3)] cells. See [http://openwetware.org/wiki/Bacterial_Transformation OWW Bacterial Transformation page] for a more general discussion of other techniques. The [http://openwetware.org/images/c/c2/Pat6709852.pdf Bloom04] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. The [http://openwetware.org/images/0/0c/Pat6960464.pdf Jesse '464 patent] describes using this buffer for DH5α cells. Unfortunately, we have found the competence of DH5α to be far lower when compared to TOP10.

Materials

Detergent-free, sterile glassware and plasticware. We use dedicated centrifuge tubes that are only used for preparation of competent cells
Table-top OD600nm spectrophotometer
SOB
CCMB80 buffer (see below)

CCMB80 buffer

10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
80 mM CaCl₂.2H₂O (11.8 g/L)
20 mM MnCl₂.4H₂O (4.0 g/L)
10 mM MgCl₂.6H₂O (2.0 g/L)
10% glycerol (100 ml/L)
adjust pH DOWN to 6.4 with 0.1N HCl if necessary
- adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
sterile filter and store at 4°C
slight dark precipitate appears not to affect its function

Procedure

Prechill plasticware and glassware

Prechill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks

Prepare TOP10 preculture by scraping a -80d°C TOP10 glycerol stock into 3ml of LB medium and shake overnight at 37°C

Preparing competent cells

Prepare TOP10 preculture by inoculating 3ml of LB medium with a -80d°C TOP10 glycerol stock into and shake overnight at 37°C
Inoculate 250 ml of SOB medium with 3 ml of preculture and grow in a 36°C shaker to an OD600nm of 0.3
- This takes approximately 2.5 hours.
- Aim for an OD600 of slightly below 0.3
Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
- Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
- It is often easier to resuspend pellets by mixing before adding large amounts of buffer
Gently resuspend in 80 ml of ice cold CCMB80 buffer
- This is is often less than completely gentle. It still works.
Incubate on ice 20 minutes
Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. Usually this additional step is not necessary.
Incubate on ice for 20 minutes
Aliquot 50 μl samples to sterile, prechilled, 1.5 ml or 2 ml eppendorf tubes and flash freeze by immersion in liquid nitrogen.
Store at -80°C indefinitely.
Test competence (see below)

Measurement of competence

Transform 50 μl of cells with 10 pg of standard pUC19
- This is 1 μl of standard pUC19 Invitrogen plasmid
Hold on ice for 30 minutes
Heat shock 60 sec at 42°C
Add 200 μl SOC
shake at 37°C for 2 hours in 14 ml aerated tubes
Plate 20 μl and 200 on AMP plates using a sterile plate spreader
- Transformation efficiency is number of colonies / (ng of DNA x partition of total volume plated) x 1000
- Competence should be at least 5x10⁶. The higher the better.

References

Hanahan91 pmid=1943786
Reusch86 pmid=3536850
Addison04 pmid=15470891
Bloom04 US Patent 6,709,852 [http://openwetware.org/images/c/c2/Pat6709852.pdf pat6709852.pdf]
Bloom05 US Patent 6,855,494 [http://openwetware.org/images/b/bd/Pat6855494.pdf pat6855494.pdf]
Jesse05 US Patent 6,960,464 [http://openwetware.org/images/0/0c/Pat6960464.pdf pat6960464.pdf]

@@ Line 59: / Line 59: @@
 ** Transformation efficiency is number of colonies / (ng of DNA x partition of total volume plated) x 1000
 ** Competence should be at least 5x10<sup>6</sup>. The higher the better.
-===5x Ligation Adjustment Buffer===
-* Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer
-* KOAc  40 mM  (40 ml/liter of 1 M KOAc solution, pH 7.0)
-* CaCl<sub>2</sub> 400 mM  (200 ml/l of a 2 M solution)
-* MnCl<sub>2</sub> 100 mM  (100 ml/l of a 1 M solution)
-* Glycerol 46.8%  (468 ml/liter)
-* pH adjustment with 2.3%  of a 10% acetic acid solution (12.8ml/liter)
-** Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --[[User:Meagan|Meagan]] 15:50, 25 January 2007 (EST)
-* water to 1 liter
-* autoclave or sterile filter
-* Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5
-*'''Reshma P. Shetty 10:49, 11 February 2008 (CST)''': Use of the ligation adjustment buffer is optional.
 ===References===