IGEM:PennState/Labbook/AudreyLeung

June 12, 2007

Primers

crp ORF (view sequence: [1])

Ggaattcgcggccgcttctagag ATGGTGCTTGGCAAACC Forward primer
TmC 60 

ctgcagcggccgctactagta TTAACGAGTGCCGTAAAC Reverse Primer
TmC 57

xylR ORF (view sequence: [2])

Ggaattcgcggccgcttctagag CCATGTTTACTAAACGTCAC Forward primer
TmC 56

ctgcagcggccgctactagta CTACAACATGACCTCGCTAT Reverse primer
TmC 58

June 26, 2007

Competent cell preparation

http://www.chem.uga.edu/scottgrp/GrpProtocols/Competent_cell_preparation.htm

There are two main methods for preparation of competent bacterial cells (14) for transformation, the calcium chloride and the electroporation method. For the calcium chloride method, a glycerol cell culture stock of the respective E. coli strain is thawed and added to 50 ml of liquid media. This culture then is preincubated at 37degC for 1 hour, transferred to an incubator-shaker, and is incubated further for 2-3 hours. The cells are pelleted by centrifugation, resuspended in calcium chloride solution, and incubated in an ice-water bath. After another centrifugation step, the resulting cell pellet again is resuspended in calcium chloride to yield the final competent cell suspension. Competent cells are stored at 4degC, for up to several days.

Calcium Chloride Protocol 1. Thaw a frozen glycerol stock of the appropriate strain of E. coli, add it to an Erlenmeyer flask containing 50 ml of pre-warmed 2xTY (1) media, and pre-incubate in a 37degC water bath for 1 hour with no shaking. Further incubate for 2-3 hours at 37degC with shaking at 250 rpm.

2. Transfer 40 ml of the cells to a sterile 50 ml polypropylene centrifuge tube, and collect the cells by centrifugation at 3000 rpm for 8 minutes at 4deg C in a GPR centrifuge (Beckman) or 6000 rpm for 8 minutes at 4degC in an RC5-B centrifuge (DuPont) equipped with an SS-34 rotor. For M13-based transformation, save the remaining 10 ml of culture in an ice-water bath for later use.

3. After centrifugation, decant the supernatant and resuspend the cell pellet in one-half volume (20 ml) of cold, sterile 50 mM calcium chloride, incubate in an ice-water bath for 20 minutes, and centrifuge as before.

4. Decant the supernatant and gently resuspend the cell pellet in one-tenth volume (4 ml) of cold, sterile 50 mM calcium chloride to yield the final competent cell suspension.

Preparation of calcium chloride competent cells for frozen storage

1. Transfer 166 ul of the competent cell suspension to sterile Falcon culture tubes.

2. Add 34 ul of sterile 100% glycerol to the 166 ul aliquots of the final competent cell suspension prepared above, giving a final concentration of 17 % glycerol.

3. The competent cells then should be placed at -70degC and can be stored indefinately.

FROZEN COMPETENT E. COLI CELLS http://www.ciwemb.edu/labs/koshland/Protocols/BACTERIA/ecolicells.html (Inoue et al., 1990 Gene 96:23)

  1. Inoculate a 5ml overnight of E.coli in LB+20 mM MgSO4.
  2. Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an A600 of 0.4-0.6. Temp is important--see original ref.
  3. Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C.
  4. Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice.
  5. Spin cells 3K, 10', 4°C.
  6. Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice.
  7. Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106 cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.

To use:

  1. To 50 or 100 ul cells, add 5-50 ng DNA. Leave 30’ on ice.
  2. Heat shock, 45 sec at 42C, then chill cells on ice about 2’.
  3. Spin down (15 sec, eppendorf fuge) and remove SN. (Removing the Manganese seems to boost efficiency about 10X) and resuspend cells in 200 ul LB.
  4. For a supercoiled plasmid, plate 1 ul of cells. For a ligation, plate 20 ul and the rest. 

TB (transformation buffer: filter sterilize and store 4°C) Product [stock] [ ]final volumes to make 100ml Pipes-NaOH pH6.7 0.5M 10 mM 2 ml CaCl2 0.5M 15 mM 3 ml KCl 2M 0.25M 12.5 ml (or 1.864g) MnCl2 1M 55 mM 5.5ml (or 1.088g) add to 100ml with ddH2O 4. To use competent cells for transformation, remove from freezer and thaw for a few minutes at 37degC. Place on ice, add plasmid DNA and incubate for one hour as in the standard transformation procedure. Then heat shock at 42degC for 2 minutes, cool briefly, add 1 ml of 2xTY and incubate for 1 hour at 37degC before spreading on plates.