Team:Amsterdam/Notebook/Protocols/Making Competent Cells

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Revision as of 11:20, 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

Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C
- room temperature works well
Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
- room temperature works well
Add glycerol to 15%
Aliquot 1 ml samples to Nunc cryotubes
Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
- This step may not be necessary
Place in -80°C freezer indefinitely.

Preparing competent cells

Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
- This takes approximately 16 hours.
- Controlling the temperature makes this a more reproducible process, but is not essential.
- Room temperature will work. You can adjust this temperature somewhat to fit your schedule
- Aim for lower, not higher OD if you can't hit this mark
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
- sometimes this is 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.
Incubate on ice for 20 minutes
Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
Store at -80°C indefinitely.
- Flash freezing does not appear to be necessary
Test competence (see below)
Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.

Measurement of competence

Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
- This is at 10 pg/μl or 10^-5 μg/μl
- This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE
Hold on ice 0.5 hours
Heat shock 60 sec at 42C
Add 250 μl SOC
Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
- using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
- For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
- Ampicillin and kanamycin appear to do fine with 1 hour growth
Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
- Good cells should yield around 100 - 400 colonies
- Transformation efficiency is (dilution factor=15) x colony count x 10⁵/µgDNA
- We expect that the transformation efficiency should be between 5x10⁸ and 5x10⁹ cfu/µgDNA

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)
CaCl₂ 400 mM (200 ml/l of a 2 M solution)
MnCl₂ 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 --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

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 23: / Line 23: @@
 ===Procedure===
-====Preparing glassware and media====
-=====Eliminating detergent=====
-Detergent is a major inhibitor of competent cell growth and transformation.  Glass and plastic
-must be detergent free for these protocols.  The easiest way to do this is to avoid washing
-glassware, and simply rinse it out.  Autoclaving glassware filled 3/4 with DI water is an effective
-way to remove most detergent residue.  Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.
 =====Prechill plasticware and glassware=====