Penn State University 2006:Preparing chemically competent cells: Difference between revisions

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''Time: O’N then 3 hr next day''
''Time: O’N then 3 hr next day''


''Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 <nowiki>"Trans. Using CaCl2"</nowiki>''
''Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 <nowiki>"Trans. Using CaCl<sub>2</sub>"</nowiki>''


Makes 64x50μL aliquots
Makes 64x50μL aliquots
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*LB
*LB
*Cells (from plate)
*Cells (from plate)
*CaCl2 Solution
*CaCl<sub>2</sub> Solution
*Dry Ice (2nd floor Althouse or S. Frear)
*Dry Ice (2nd floor Althouse or S. Frear)


===<font color="red">Pre</font>===  
===<font color="red">Pre</font>===  
*Prepare CaCl2 solution<sup>1</sup>
*Prepare CaCl<sub>2</sub> solution<sup>1</sup>
*Autoclave centrifuge bottles/tubes and chill on ice
*Autoclave centrifuge bottles/tubes and chill on ice
*Before centrifuge steps, make sure centrifuge is on and at 0°C
*Before centrifuge steps, make sure centrifuge is on and at 0°C
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#Ice for 10 min.
#Ice for 10 min.
#Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
#Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
#Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl2 buffer.
#Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl<sub>2</sub> buffer.
#Spin 1100g/5 min/0°C.   
#Spin 1100g/5 min/0°C.   
#Gently resuspend pellet in 8 mL ice-cold CaCl2.  Let stand on ice for 30 min.
#Gently resuspend pellet in 8 mL ice-cold CaCl<sub>2</sub>.  Let stand on ice for 30 min.
#Spin 1100g/5 min/0°C.  Decant supernatant & resuspend <u>''well''</u> in 1.6 mL ice-cold CaCl2.  Cells may stand on ice for 12-24 hrs to increase competency.   
#Spin 1100g/5 min/0°C.  Decant supernatant & resuspend <u>''well''</u> in 1.6 mL ice-cold CaCl<sub>2</sub>.  Cells may stand on ice for 12-24 hrs to increase competency.   
#Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs.  Immerse immediately in crushed dry ice.
#Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs.  Immerse immediately in crushed dry ice.
#Transfer to box(es) and store in -80°C freezer.
#Transfer to box(es) and store in -80°C freezer.
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<sup>1</sup>CaCl2(per L)
<sup>1</sup>CaCl<sub>2</sub>(per L)
*20 g bactotryptone
*20 g bactotryptone
*5 g bacto-yeast extract
*5 g bacto-yeast extract
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*Adjust to pH 7.0 w/ NaOH
*Adjust to pH 7.0 w/ NaOH
*Autoclave
*Autoclave
*Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)
*Add filter-sterilized MgCl<sub>2</sub>, MgSO<sub>4</sub> solution to give final Mg<sup>2+</sup> conc. of 20 mM (i.e. 10 mM MgCl<sub>2</sub>, 10 mM MgSO<sub>4</sub>)




<sup>2</sup>Procedure can be scaled up or down as necessary
<sup>2</sup>Procedure can be scaled up or down as necessary

Revision as of 19:32, 23 October 2006

Making Cells Competent

Time: O’N then 3 hr next day

Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 "Trans. Using CaCl<sub>2</sub>"

Makes 64x50μL aliquots

Media/Reagents

  • LB
  • Cells (from plate)
  • CaCl2 Solution
  • Dry Ice (2nd floor Althouse or S. Frear)

Pre

  • Prepare CaCl2 solution1
  • Autoclave centrifuge bottles/tubes and chill on ice
  • Before centrifuge steps, make sure centrifuge is on and at 0°C

Competent Cells: Day 0

  1. Streak cells on LB plate
  2. Grow overnight at 37°C

Competent Cells: Day 1 (optional)

  1. Inoculate overnight starter culture (2 mL) w/ colony from plate

Competent Cells: Day 2

  1. Inoculate 80 mL2 with appropriate amount of O’N to obtain OD6000~0.1. Place cells on ice at OD600=0.375 (do not allow growth past an OD of 0.4, as this decreases subsequent transformation efficiency). Typically this will take 40-60 min (check every 5 min when OD is getting near 0.375).
  2. Ice for 10 min.
  3. Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
  4. Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl2 buffer.
  5. Spin 1100g/5 min/0°C.
  6. Gently resuspend pellet in 8 mL ice-cold CaCl2. Let stand on ice for 30 min.
  7. Spin 1100g/5 min/0°C. Decant supernatant & resuspend well in 1.6 mL ice-cold CaCl2. Cells may stand on ice for 12-24 hrs to increase competency.
  8. Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs. Immerse immediately in crushed dry ice.
  9. Transfer to box(es) and store in -80°C freezer.



1CaCl2(per L)

  • 20 g bactotryptone
  • 5 g bacto-yeast extract
  • 0.5 g NaCl
  • 0.19 g KCl
  • Adjust to pH 7.0 w/ NaOH
  • Autoclave
  • Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)


2Procedure can be scaled up or down as necessary

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