Wiese Lab:Competent Cell Prep: Difference between revisions
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==RbCl Competent Cell Prep== | ==RbCl Competent Cell Prep== | ||
====Solutions and Supplies==== | ====Solutions and Supplies==== | ||
Prepare RF1 and RF2 and store at 4oC. Stable for > 1yr | |||
*sterilized 250 ml centrifuge bottles <br> | |||
*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) | |||
* Prepare RF1 and RF2 and store at 4oC. Stable for > 1yr: | |||
=====RF1===== | =====RF1===== | ||
{| {{table}} | {| {{table}} | ||
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*Adjust final pH to 6.8 using NaOH. Filter-sterilize. | *Adjust final pH to 6.8 using NaOH. Filter-sterilize. | ||
====Cell Preparation Procedure==== | ====Cell Preparation Procedure==== |
Latest revision as of 16:31, 26 February 2008
RbCl Competent Cell Prep
Solutions and Supplies
- 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)
- Prepare RF1 and RF2 and store at 4oC. Stable for > 1yr:
RF1
Combine for 1 L: | [final] | |
RbCl | 12 g | 100 mM |
MnCl2 4H2O | 9.9 g | 50 mM |
K acetate | 30 ml of 1M stock, pH 7.5 | 35 mM |
CaCl2 2H2O | 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
Combine for 1 L: | [final] | |
MOPS | 10 mM | |
RbCl | 1.2 g | 10 mM |
CaCl2 2H2O | 11 g | 75 mM |
Glycerol | 150 g | 15% wt/vol |
- Adjust final pH to 6.8 using NaOH. Filter-sterilize.
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 pDNA | 2 ng/ul |
vol used | 1 ul |
total amt pDNA | 2 ng |