Wiese Lab:Competent Cell Prep

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==RbCl Competent Cell Prep==
 +
====Solutions and Supplies====
 +
Prepare RF1 and  RF2 and store at 4oC.  Stable for > 1yr.
 +
=====RF1=====
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Combine for 1 L:'''
 +
| align="center" style="background:#f0f0f0;"|
 +
| align="center" style="background:#f0f0f0;"|'''[final]'''
 +
|-
 +
| RbCl||12 g||100 mM
 +
|-
 +
| MnCl<sub>2</sub> 4H<sub>2</sub>O||9.9 g||50 mM
 +
|-
 +
| K acetate||30 ml of 1M stock, pH 7.5||35 mM
 +
|-
 +
| CaCl<sub>2</sub> 2H<sub>2</sub>O||1.5 g||10 mM
 +
|-
 +
| Glycerol||150 g||15% wt/vol
 +
|}
 +
*Adjust final pH to 5.8 using 0.2M acetic acid.  Filter-sterilize.
 +
=====RF2=====
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Combine for 1 L:'''
 +
| align="center" style="background:#f0f0f0;"|
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| align="center" style="background:#f0f0f0;"|'''[final]'''
 +
|-
 +
| MOPS||||10 mM
 +
|-
 +
| RbCl||1.2 g||10 mM
 +
|-
 +
| CaCl<sub>2</sub> 2H<sub>2</sub>O||11 g||75 mM
 +
|-
 +
| Glycerol||150 g||15% wt/vol
 +
|}
 +
*Adjust final pH to 6.8 using NaOH.  Filter-sterilize.
 +
 +
*sterilized 250 ml centrifuge bottles <br>
 +
*sterilized 1.5 ml microfuge tubes
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*300 ml LB in 2L flask
 +
*100 ml LB in 1L flask
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*100 ml chilled RF1 per 300 ml ''E. coli'' culture (or 33 ml per 100 ml culture)
 +
*24 ml chilled RF2 per 300 ml ''E. coli'' culture (or8 ml per 100 ml culture)
 +
 +
====Cell Preparation Procedure====
 +
=====Prepare competent bacterial cells using RbCl2=====
 +
::-  A 300-ml culture will yield ~ 60 aliquots of 400 ul competent cells
 +
::-  A 100-ml culture will yield ~ 20 aliquots of 400 ul competent cells
 +
 +
*Streak DH5alpha from frozen glycerol stock.
 +
*Prepare a 2 ml culture using a single CFU and 2 ml L Broth. 
 +
*Shake at 37C O/N.
 +
 +
=====Next a.m.=====
 +
*Inoc 300 ml sterile LB  in 2L flask using 300 ul of O/N culture.  (1:1000 dilution)
 +
*Monitor OD 550 from initial until 0.2 to 0.6.  [0.4 to 0.55 optimum]
 +
*Transfer culture to centrifuge bottle and chill on ice 10 -15 min.
 +
*Pellet cells by centrifugation at 3000 rpm 12-15 min at 4C.
 +
*Decant liquid and resuspend in 1/3 original volume (100 ml or 33 ml) chilled RF1 buffer.
 +
*Optimally, resuspend using a 25-ml disposable pipet.
 +
**RbCl will permanently stain glass pipets.
 +
**Note:  resuspend gently, DO NOT VORTEX. 
 +
***Want to maintain pili structures on surface of E coli cell.
 +
*Continue mixing until cells are evenly resuspended and no clumps are visible.
 +
*Incubate cells/RbCl buffer 1 on ice for 45min to 60 min.
 +
*Pellet cells by centrifugation at 3000 rpm 12-15 min at 4C.
 +
*Decant liquid and gently resuspend in 1/12.5 original volume (24 ml or 8 ml) chilled RF2 buffer.
 +
*Incubate cells/RbCl buffer 2 on ice for 15min.
 +
*Distribute 400 ul into chilled 1.5 ml microfuge tubes and freeze on dry ice.
 +
*Store at -80C.
 +
 +
=====Determine transformation efficiency=====
 +
*Dilute control plasmid DNA (known DNA conc) to 2 ng/ul and transform using 1 ul.
 +
*Thaw competent cells on ice. 
 +
*Compare previous lot to current lot
 +
*Combine 1ul of diluted pDNA and 200 ul competent cells.
 +
*Incubate on ice 30-60  min (40 min).
 +
*Heat shock at 42C for 1 min, place on ice 5 - 15 min.
 +
*Add 500 ul LB and incubate at 37C for 30-60 min (45 min).
 +
*Plate 10 ul and 100 ul onto antibiotic plate.
 +
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Calculate transformation efficiency'''
 +
| align="center" style="background:#f0f0f0;"|'''For example'''
 +
|-
 +
| conc pDNA||2 ng/ul
 +
|-
 +
| vol used||1 ul
 +
|-
 +
| total amt pDNA||2 ng
 +
|}

Revision as of 19:11, 26 February 2008

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Contents

RbCl Competent Cell Prep

Solutions and Supplies

Prepare RF1 and RF2 and store at 4oC. Stable for > 1yr.

RF1
Combine for 1 L: [final]
RbCl12 g100 mM
MnCl2 4H2O9.9 g50 mM
K acetate30 ml of 1M stock, pH 7.535 mM
CaCl2 2H2O1.5 g10 mM
Glycerol150 g15% wt/vol
  • Adjust final pH to 5.8 using 0.2M acetic acid. Filter-sterilize.
RF2
Combine for 1 L: [final]
MOPS10 mM
RbCl1.2 g10 mM
CaCl2 2H2O11 g75 mM
Glycerol150 g15% wt/vol
  • Adjust final pH to 6.8 using NaOH. Filter-sterilize.
  • sterilized 250 ml centrifuge bottles
  • sterilized 1.5 ml microfuge tubes
  • 300 ml LB in 2L flask
  • 100 ml LB in 1L flask
  • 100 ml chilled RF1 per 300 ml E. coli culture (or 33 ml per 100 ml culture)
  • 24 ml chilled RF2 per 300 ml E. coli culture (or8 ml per 100 ml culture)

Cell Preparation Procedure

Prepare competent bacterial cells using RbCl2
- A 300-ml culture will yield ~ 60 aliquots of 400 ul competent cells
- A 100-ml culture will yield ~ 20 aliquots of 400 ul competent cells
  • Streak DH5alpha from frozen glycerol stock.
  • Prepare a 2 ml culture using a single CFU and 2 ml L Broth.
  • Shake at 37C O/N.
Next a.m.
  • Inoc 300 ml sterile LB in 2L flask using 300 ul of O/N culture. (1:1000 dilution)
  • Monitor OD 550 from initial until 0.2 to 0.6. [0.4 to 0.55 optimum]
  • Transfer culture to centrifuge bottle and chill on ice 10 -15 min.
  • Pellet cells by centrifugation at 3000 rpm 12-15 min at 4C.
  • Decant liquid and resuspend in 1/3 original volume (100 ml or 33 ml) chilled RF1 buffer.
  • Optimally, resuspend using a 25-ml disposable pipet.
    • RbCl will permanently stain glass pipets.
    • Note: resuspend gently, DO NOT VORTEX.
      • Want to maintain pili structures on surface of E coli cell.
  • Continue mixing until cells are evenly resuspended and no clumps are visible.
  • Incubate cells/RbCl buffer 1 on ice for 45min to 60 min.
  • Pellet cells by centrifugation at 3000 rpm 12-15 min at 4C.
  • Decant liquid and gently resuspend in 1/12.5 original volume (24 ml or 8 ml) chilled RF2 buffer.
  • Incubate cells/RbCl buffer 2 on ice for 15min.
  • Distribute 400 ul into chilled 1.5 ml microfuge tubes and freeze on dry ice.
  • Store at -80C.
Determine transformation efficiency
  • Dilute control plasmid DNA (known DNA conc) to 2 ng/ul and transform using 1 ul.
  • Thaw competent cells on ice.
  • Compare previous lot to current lot
  • Combine 1ul of diluted pDNA and 200 ul competent cells.
  • Incubate on ice 30-60 min (40 min).
  • Heat shock at 42C for 1 min, place on ice 5 - 15 min.
  • Add 500 ul LB and incubate at 37C for 30-60 min (45 min).
  • Plate 10 ul and 100 ul onto antibiotic plate.
Calculate transformation efficiency For example
conc pDNA2 ng/ul
vol used1 ul
total amt pDNA2 ng
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