Difference between revisions of "Rubidiumchlorid competent E.coli cells"
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'''!!!Important!!!''' | '''!!!Important!!!''' | ||
− | For making Rubidiumchlorid competent E.coli cells you already need one sample of competent cells frozen at - | + | For making Rubidiumchlorid competent ''E.coli'' cells you already need one sample of competent cells frozen at -70°C. |
These cell are used for the [[E coli Chemical Transformation]] protocol. | These cell are used for the [[E coli Chemical Transformation]] protocol. | ||
− | == Generation - | + | == Generation -70°C Rubidiumchlorid competent E.coli cells stocks for chemical transformation == |
This is a protocol for 200ml medium which yields enough cells for 100 to 200 transformation. If cells for fewer transformations are needed feel free to scale down. | This is a protocol for 200ml medium which yields enough cells for 100 to 200 transformation. If cells for fewer transformations are needed feel free to scale down. | ||
− | # Prepare 2 ml preculture with - | + | # Prepare 2 ml preculture with -70°C competent cells in [[LB]] and grow cells by shaking at 37°C for 14 hrs. |
# Dilute 2 ml of fresh O/N culture in 200 ml LB + add 2 ml of 1M MgCl<sub>2</sub> and 1M MgSO<sub>4</sub> to get to a final concentration of 10 mM and grow cells until they reach the OD of 0,6-0,7 at 600 nm. | # Dilute 2 ml of fresh O/N culture in 200 ml LB + add 2 ml of 1M MgCl<sub>2</sub> and 1M MgSO<sub>4</sub> to get to a final concentration of 10 mM and grow cells until they reach the OD of 0,6-0,7 at 600 nm. | ||
#:'''CRITICAL STEP: From here cells should be kept at 4°C, so pre-cool centrifuge etc!''' | #:'''CRITICAL STEP: From here cells should be kept at 4°C, so pre-cool centrifuge etc!''' |
Latest revision as of 16:20, 26 March 2024
!!!Important!!! For making Rubidiumchlorid competent E.coli cells you already need one sample of competent cells frozen at -70°C.
These cell are used for the E coli Chemical Transformation protocol.
Generation -70°C Rubidiumchlorid competent E.coli cells stocks for chemical transformation
This is a protocol for 200ml medium which yields enough cells for 100 to 200 transformation. If cells for fewer transformations are needed feel free to scale down.
- Prepare 2 ml preculture with -70°C competent cells in LB and grow cells by shaking at 37°C for 14 hrs.
- Dilute 2 ml of fresh O/N culture in 200 ml LB + add 2 ml of 1M MgCl2 and 1M MgSO4 to get to a final concentration of 10 mM and grow cells until they reach the OD of 0,6-0,7 at 600 nm.
- CRITICAL STEP: From here cells should be kept at 4°C, so pre-cool centrifuge etc!
- Place the cells on ice for 20 min and cool down the glaspipettes and Eppis at -20°C.
- Spin down the cells for 8 min at 4500g at 4°C. Remove supernatant.
- Resuspend the cells in 66 ml (1/3 of the volume) precooled RFI-solution at 0°C on ice and leave them for 30 min in the cold room.
- Spin down the cells for 8 min at 3000 rpm at 4°C.Remove supernatant.
- Resuspend cells in 16 ml of precooled RFII-solution and leave them for 30 min on 0°C.
- Freeze cells in 50 or 100 µl Aliquots with liquid Nitrogen.
- Store aliquots at -80°C immediately.
- Cryopreserved cells can be stored at -80°C for at least 12 months.
- To use, thaw the cells on ice.
RFI Solution (RFI)
Storage of RFI and RFII should be always at 4°C.
Ingredient | Amount per 500 ml | Final concentration |
---|---|---|
Rubidiumchlorid | 6.0 g | 100 mM |
MnCl2 x 4H2O | 4.95 g | 50 mM |
Potassium Acetate (1 Mol pH 7,5 ) | 15.0 ml | 30 mM |
CaCl2 x 2H2O | 0.75 g (2%) | 10 mM |
Glycerol | 75.0 g | 15 % |
To adjust the pH of RFI use 0,2 M acetic acid and set the pH to 5,8. Afterwards the solution has to be filtered sterilly.
Procedure of making 1 M pH 7,5 Potassium Acetate:
- Weigh in 9,81 g and dilute it in 100 ml H2O.
- To adjust the pH use glacial acetic acid. Be careful, one drop is enough.
RFII Solution (RFII)
Ingredient | Amount per 500 ml | Final concentration |
---|---|---|
MOPs 0,5 M stock | 10 ml | 10 mM |
Rubidiumchlorid | 0.6 g | 10 mM |
CaCl2 x 2H2O | 5.50 g (2%) | 75 mM |
Glycerol | 75.0 g | 15 % |
To adjust the pH of RFII use NaOH and set the pH to 5,8. Afterwards the solution has to be filtered sterilly.
Procedure of making 0,5 M pH 6,8 MOPs:
- Weigh in 10,46 g and dilute it in 100 ml H2O.
- To adjust the pH use NaOH.