Team:Amsterdam/Notebook/Protocols/Making Competent Cells

From 2011.igem.org

(Difference between revisions)
(→Procedure)
(→Preparing seed stocks)
Line 28: Line 28:
====Preparing seed stocks====
====Preparing seed stocks====
-
* Streak TOP10 cells on an [[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]] plate and grow for single colonies at 23°C
+
* Prepare TOP10 preculture by scraping a -80d⪚C TOP10 glycerol stock into 3ml of [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Preparing_Media LB] medium
** room temperature works well
** room temperature works well
* Pick single colonies into 2 ml of SOB medium  and shake overnight at 23°C
* Pick single colonies into 2 ml of SOB medium  and shake overnight at 23°C

Revision as of 11:24, 19 September 2011

Contents

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
    • 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

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

Retrieved from "http://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Making_Competent_Cells"