Silver: LiOAc Transformation: Difference between revisions
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==Materials== | |||
*50% Polyethylene glycol | |||
**100 g (3350 g/mol) PEG | |||
**Add ddH<sub>2</sub>O to 200 ml | |||
**Mix with heat and filter (takes a while) | |||
*10x TE/LiOAc | |||
**20 ml 1 M Tris-HCl (100 mM final) | |||
**4 ml 0.5 M EDTA (10 mM final) | |||
**20.4 g LiOAc | |||
**176 ml ddH<sub>2</sub>O | |||
**Filter | |||
*40% PEG / 1xTE/LiOAc | |||
**10 ml 10x TE/LiOAc | |||
**80 ml 50% PEG | |||
**10 ml ddH<sub>2</sub>O | |||
**Mix and filter (takes a while) | |||
*10 mg/ml salmon sperm DNA (preboiled for 10 min) | |||
*DMSO | |||
*Sterile/autoclaved glass spreading beads | |||
*Selection plates (depends on experiment) | |||
**YEPD/G418 selection plates | |||
**#Mix the following ingredients in a 2 liter flask | |||
**#*10 g yeast extract | |||
**#*20 g bactopeptone | |||
**#*20 g dextrose (glucose) | |||
**#*20 g bactoagar | |||
**#*1 L ddH<sub>2</sub>O | |||
**#*stir bar | |||
**#Autoclave on a liquid cycle for 45 min | |||
**#Cool on a stir plate | |||
**#Add 200 µl of 1 g/ml G418 just before pouring into plates | |||
**#*Final G418 concentration is 200 µg/ml | |||
==Protocol== | |||
#Grow cells to 1-2 x 10<sup>7</sup> cells/ml -- 10 ml per transformation | |||
#Spin down cells 2 min / 2300 rpm | |||
#Resuspend in ddH<sub>2</sub>O (10 ml per transformation), spin down again | |||
#Resuspend in 1 ml ddH<sub>2</sub>O and spin 10 sec at 14,000 rpm in microfuge | |||
#Wash twice with 1 ml 1x TE/LiOAc, spin (can be stored at 4°C for a week for plasmid transformations, not recommended for integrations), if you use immediately resuspend cells in 50 ul 1x TE/LiOAc | |||
#Boil tube of 10 mg/ml salmon sperm DNA for 5 min, cool on ice | |||
#For transformations add | |||
#*2.5 µl ssDNA | |||
#*Transforming DNA (usually do three amounts) | |||
#*50 µl cells in 1x TE/LiOAc | |||
#Add 300 µl of 40% PEG4000 (3350) / 1xTE/LiOAc, vortex well | |||
#Incubate at 30°C on roller for 30 min (25°C for ''ts'' strains) | |||
#Add 35 µl DMSO and vortex well | |||
#Heat shock for 15 min at 42°C (15 min at 37°C or 25°C for ''ts'' strains) | |||
#Pellet cells and resuspend in 150 µl ddH<sub>2</sub>O and spread all onto one plate (unless transforming with a plasmid, only do 1/10th the amount then), spread with sterile/autoclaved glass beads | |||
#Incubate overnight at 25°C (''ts'' strains) or 30°C on a selective media plate | |||
#*For G418 resistance you need to let the cells recover, do either of these two options: | |||
#*#Plate on YEPD and grow overnight then replica-plate onto YEPD/G418 the next day | |||
#*#Inoculate YEPD liquid and let grow for 4 h at 30°C, then plate onto YEPD/G418 |
Latest revision as of 14:17, 20 September 2005
Back to Silver Lab
Back to Protocols
Materials
- 50% Polyethylene glycol
- 100 g (3350 g/mol) PEG
- Add ddH2O to 200 ml
- Mix with heat and filter (takes a while)
- 10x TE/LiOAc
- 20 ml 1 M Tris-HCl (100 mM final)
- 4 ml 0.5 M EDTA (10 mM final)
- 20.4 g LiOAc
- 176 ml ddH2O
- Filter
- 40% PEG / 1xTE/LiOAc
- 10 ml 10x TE/LiOAc
- 80 ml 50% PEG
- 10 ml ddH2O
- Mix and filter (takes a while)
- 10 mg/ml salmon sperm DNA (preboiled for 10 min)
- DMSO
- Sterile/autoclaved glass spreading beads
- Selection plates (depends on experiment)
- YEPD/G418 selection plates
- Mix the following ingredients in a 2 liter flask
- 10 g yeast extract
- 20 g bactopeptone
- 20 g dextrose (glucose)
- 20 g bactoagar
- 1 L ddH2O
- stir bar
- Autoclave on a liquid cycle for 45 min
- Cool on a stir plate
- Add 200 µl of 1 g/ml G418 just before pouring into plates
- Final G418 concentration is 200 µg/ml
- Mix the following ingredients in a 2 liter flask
- YEPD/G418 selection plates
Protocol
- Grow cells to 1-2 x 107 cells/ml -- 10 ml per transformation
- Spin down cells 2 min / 2300 rpm
- Resuspend in ddH2O (10 ml per transformation), spin down again
- Resuspend in 1 ml ddH2O and spin 10 sec at 14,000 rpm in microfuge
- Wash twice with 1 ml 1x TE/LiOAc, spin (can be stored at 4°C for a week for plasmid transformations, not recommended for integrations), if you use immediately resuspend cells in 50 ul 1x TE/LiOAc
- Boil tube of 10 mg/ml salmon sperm DNA for 5 min, cool on ice
- For transformations add
- 2.5 µl ssDNA
- Transforming DNA (usually do three amounts)
- 50 µl cells in 1x TE/LiOAc
- Add 300 µl of 40% PEG4000 (3350) / 1xTE/LiOAc, vortex well
- Incubate at 30°C on roller for 30 min (25°C for ts strains)
- Add 35 µl DMSO and vortex well
- Heat shock for 15 min at 42°C (15 min at 37°C or 25°C for ts strains)
- Pellet cells and resuspend in 150 µl ddH2O and spread all onto one plate (unless transforming with a plasmid, only do 1/10th the amount then), spread with sterile/autoclaved glass beads
- Incubate overnight at 25°C (ts strains) or 30°C on a selective media plate
- For G418 resistance you need to let the cells recover, do either of these two options:
- Plate on YEPD and grow overnight then replica-plate onto YEPD/G418 the next day
- Inoculate YEPD liquid and let grow for 4 h at 30°C, then plate onto YEPD/G418
- For G418 resistance you need to let the cells recover, do either of these two options: