Team:METU Turkey/Results Discussion/Competent Cell Preparation

From 2010.igem.org

MAIN STEPS/TIME TABLE
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- Incubation [12-14 h] >>> Spread plate
- Incubation [12-14 h] >>> Liquid culture
- Competent cell preparation

MATERIALS
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- Centrifuge
- Autoclave
- P1000, P10 and P2
- Incubator with shaker [M15]
- pH meter [M08]

- Stock competent cells
- LB
- 2 mL epp
- Strep
- Ice
- Liquid nitrogen
- Bacto yeast extract
- Bacto tryptone
- Magnesium sulfate
- Potassium hydroxide
- Potassium acetate
- Rubidium chloride
- Calcium chloride
- Manganese chloride
- Glycerol
- Dilute acetic acid
- Filter
- MOPS
- Dilute NaOH

CHECK-LIST PROCEDURE
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- Inoculate streak plates from liquid stock competent cells and incubate overnight at 37 C
- Put 10 mL LB + strep + colony into 3 eppendorf. Incubate overnight at 37 C (- Inoculate 200 ul to 1000 ul from overnight culture into 100-500 ml Psi broth (scale up or down as needed). Incubate at 37 C with aeration to A600=0.6-0.7
- Ice 15 min. From this step onward the cells must remain COLD. (4C or on ice)
- Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)
- Discard supernatant and add 0.4 volume (ie of original volume, here it is 40-400 ml) TfbI, resuspend and ice 15 min.
- Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)
- Discard supernatant and resuspend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at
-70C for storage. I usually save these in 100ul to 200ul aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.

SOLUTIONS
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Preparation of Psi broth (per liter)
- 5 g bacto yeast extract
- 20 g bacto tryptone
- 5 g magnesium sulfate
- PH 7.6 with potassium hydroxide
- Autoclave 40 min

Preparation of TfbI (per 200 ml)
- 0.588 g potassium acetate (final molarity/conc= 30 mM)
- 2.42 g rubidium chloride (final molarity/conc= 100 mM)
- 0.294 g calcium chloride (final molarity/conc= 10 mM)
- 2.0 g manganese chloride (final molarity/conc= 50 mM)
- 30 mL glycerol (15% v/v)
- Adjust PH 5.8 with dilute acetic acid
- Sterilize with filter

Preparation of TfbII (per 100 ml)
- 0.21 g MOPS (final molarity/conc= 10 mM)
- 1.1 g calcium chloride (final molarity/conc= 75 mM)
- 0.121 g rubidium chloride (final molarity/conc= 10 mM)
- 15 mL glycerol (15% v/v)
- Adjust PH 6.5 with dilute NaOH
- Sterilize with filter

NOTES
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typically transform 50-100 ul cells with 2-10 ul of a ligation reaction, and you should get between 1x10exp8 to 1x10exp9 cfu's/ug DNA.

Retrived from http://130.15.90.245/e__coli_competent_cells.htm

- All steps should be done in ice.
- Pellet centrifuge steplerinden sonra, once bir miktar TFB solution ile suspend edilmeli sonra gereken degerde dilute edilmeli for enhance the bacterial yield.
- 3 ayri kapta hazirlanacak bacterial culture lar icin ilk once totalde culture hazirlanmali sonra bolusturulmeli for control the total bacterial yield.
- Aeration of culture is critical for competence. For 200mL or 100mL culture keep in 1000mL flask.
- 600 nm deki absorbance olcumlerinde bacterial growth absorbance inin 0.6 olmasi icin gereken zamani yaklasik olarak tayin etmek icin Hamdi Hoca bir equation onerdi
ln(A2)- ln(A1) = k. t(min)
A2= Last measured absorbance value
A1= Previous measured absorbance value
t= time between these two measurements

For medium and buffer preparations different from above procedure we prepared Psi Broth 600mL
- 3 g bacto yeast extract
- 12 g bacto tryptone
- 6 g magnesium sulfate

TfbI 250 ml
- 0.705 g potassium acetate (final molarity/conc= 30 mM)
- 2.9 g rubidium chloride (final molarity/conc= 100 mM)
- 0.352 g calcium chloride (final molarity/conc= 10 mM)
- 3.3 g manganese chloride (final molarity/conc= 50 mM)
- 36 mL glycerol (15% v/v)

TfbII per 30 ml
- 0.063 g MOPS (final molarity/conc= 10 mM)
- 0.33 g calcium chloride (final molarity/conc= 75 mM)
- 0.0363 g rubidium chloride (final molarity/conc= 10 mM)
- 4.5 mL glycerol (15% v/v)