"
Team:CU-Boulder/Notebook/Protocols
From 2014.igem.org
(→Bacterial Conjugation) |
(→Making Chemically Competent Bacteria) |
||
| Line 106: | Line 106: | ||
'''To prepare for Day 2''' | '''To prepare for Day 2''' | ||
| - | + | * *Set centrifuge to 4°C | |
| - | + | * *MgCl2 and CaCl2 solutions | |
| - | + | * *Thaw DMSO | |
| - | + | * *Chill tubes | |
| - | + | * *Acquire liquid nitrogen or dry ice | |
'''Day 1''' | '''Day 1''' | ||
| Line 116: | Line 116: | ||
'''Day 2''' | '''Day 2''' | ||
| - | + | *1.Add 0.5mL of the overnight culture to 50mL LB | |
| - | + | *2.Grow until OD is between 0.2 and 0.4 | |
| - | + | *3.Incubate on ice for 30 minutes | |
| - | + | *4.Centrifuge for 10 minutes at 2700 x g and 4°C | |
| - | + | *5.Decant. Dry upside down on a paper towel for 1 minute | |
| - | + | *6.Completely resuspend in 30mL 0.8M MgCl2, 0.2M CaCl2 | |
**a. Gently vortex | **a. Gently vortex | ||
| - | + | *7.Centrifuge for 10 minutes at 2700 x g and 4°C | |
| - | + | *8.Decant. Dry upside down on a paper towel for 1 minute | |
| - | + | *9.Fully resuspend in 2mL of 0.1M CaCl2 | |
| - | + | *10.Chill sample on ice. Add 70ul DMSO, keeping the sample tube on ice | |
| - | + | *11.Swirl to mix | |
| - | + | *12.Incubate on ice for 15 minutes | |
| - | + | *13.Add 70ul DMSO, swirl to mix, keeping the sample tube on ice | |
| - | + | *14.Dispense 200ul into pre-chilled 1.7mL tubes | |
| - | + | *15.Snap freeze with liquid nitrogen or dry ice | |
| - | + | *16.Store at -80°C until ready to use | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
==Isolation of single-stranded phagemid DNA using M13K07 Helper Phage== | ==Isolation of single-stranded phagemid DNA using M13K07 Helper Phage== | ||
Revision as of 03:23, 4 October 2014
Amplification of Phage using Helper Phage
Need
- Plate of infectable cells that contain F’ episome
- 2.5M NaCl/20% PEG-8000
- 1x TBS
Day 1
- 1.Add a fresh colony of infectable cells to 50mL LB in 125mL flask.
- a. Include phagemid antibiotic only.
- b. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
- 2.Add the helper phage to a final concentration of 1 x10^8 phage/mL
- 3.Incubate for 60-90 minutes, shaking
- 4.Add Helper Phagemid antibiotic to a high concentration
- 5.Grow for 14-18 hours at 37°C, shaking
Day 2
- Spin culture at 4,000 x g for 10 minutes
- Transfer supernatant to a fresh conical. Repeat spin on supernatant
- Transfer the upper 90% of supernatant to a new conical
- Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix
- Incubate at 4°C for at least 60 minutes
- Centrifuge at 12,000 x g for 10 minutes. Carefully decant
- Spin again briefly
- Gently resuspend pellet in 1.6mL 1x TBS
- Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes
- Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes
- Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each
- Let sit at room temperature for 5 minutes
- Spin at 1300 x g for 10 minutes
- Decant the supernatant
- Spin briefly. Remove supernatant with pipet
- Resuspend pellet in 200ul 1x TBS.
- a. If desired, combine contents of both tubes into one
2.5M NaCl/20% PEG-8000 (5x)
- 100 g PEG-8000
- 75 g NaCl
- 400 mL H2O
- Bring final volume to 500 mL
TBS (1x)
- 6.05 g Tris
- 8.76 g NaCl
- 800 mL H2O
- Adjust pH to 7.6 with 1M HCL
- Adjust volume to 1 L
Bacterial Transformation Using Frozen Competent Cells
Before you start
- Heat hot plate or water bath to 42°C
- Warm selection plates to 37°C
Transformation
- 1.Thaw chemically competent cells on ice for 10-15 minutes
- 2.Add 40ul cells to fresh 1.7mL tube
- 3.Add DNA
- a. If using a ligation product add up to 10ul of sample
- b. If using supercoiled plasmid add 100ng
- 4.Incubate on ice for 30 minutes
- 5.Heat shock cells on hot plate (or water bath) for 30-45s* @ 42°C
- 6.Incubate on ice for 2-5 minutes
- 7.Add 200 μL SOC and shake gently for 1-2 hours @ 37° C
- a.(Note: Can also recover in 960ul. After recovery, gently spin cells and remove supernatant. Resuspend in 200ul LB)
- 8.Plate 100-200ul cells onto selection plates
- a. If high efficiency is expected, we suggest also plating a 1:10 dilution
- 9.Once dry, turn upside down (agar on top) and incubate overnight @ 37° C
- *Optimal timing depends on cells
SOC (1L)
- 20 g Tryptone
- 5 g YeastExtract
- 4.8 g MgSO4
- 3.6 g Dextrose
- 0.5 g NaCl
- 0.186 g KCL
Bacterial Conjugation
Need
- Donor cells: Cells already containing F’ episome
- Recipient cells: Cells with a resistance marker that is absent from donor cells
- Double selection plate containing antibiotic to select for F’ episome and a second antibiotic to select for the recipient cells
Day 1
- Set up liquid overnight of donor cells. Include antibiotic
- Set up liquid overnight of recipient cells. Include antibiotic
Day 2
- 1.Mix 500ul of each overnight sample in a new tube. Mix by pipetting or flicking
- 2.Incubate for 30 minutes at 37°C, shaking
- 3.Plate 100ul onto double selection plate
- a.We advise also plating a 1:10 dilution
- 4.Incubate at 37°C
http://inst.bact.wisc.edu/inst/index.php?module=Book&func=displayarticle&art_id=128
Making Chemically Competent Bacteria
To prepare for Day 2
- *Set centrifuge to 4°C
- *MgCl2 and CaCl2 solutions
- *Thaw DMSO
- *Chill tubes
- *Acquire liquid nitrogen or dry ice
Day 1
- Grow cells O/N
Day 2
- 1.Add 0.5mL of the overnight culture to 50mL LB
- 2.Grow until OD is between 0.2 and 0.4
- 3.Incubate on ice for 30 minutes
- 4.Centrifuge for 10 minutes at 2700 x g and 4°C
- 5.Decant. Dry upside down on a paper towel for 1 minute
- 6.Completely resuspend in 30mL 0.8M MgCl2, 0.2M CaCl2
- a. Gently vortex
- 7.Centrifuge for 10 minutes at 2700 x g and 4°C
- 8.Decant. Dry upside down on a paper towel for 1 minute
- 9.Fully resuspend in 2mL of 0.1M CaCl2
- 10.Chill sample on ice. Add 70ul DMSO, keeping the sample tube on ice
- 11.Swirl to mix
- 12.Incubate on ice for 15 minutes
- 13.Add 70ul DMSO, swirl to mix, keeping the sample tube on ice
- 14.Dispense 200ul into pre-chilled 1.7mL tubes
- 15.Snap freeze with liquid nitrogen or dry ice
- 16.Store at -80°C until ready to use
Isolation of single-stranded phagemid DNA using M13K07 Helper Phage
Need
- Fresh plate of infectable cells (contain F’ episome)
- 2.5M NaCl/20% PEG-8000
- TBS, TE, phenol, phenol/chloroform, chloroform
Day 1
1. Add a fresh colony to 50mL LB in 125mL flask. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
2. Add M13KO7 helper phage to a final concentration of 1 x10^8 phage/mL*
3. Continue shaking for 60-90 minutes
4. Add Kanamycin to final concentration of 70ug/mL
5. Grow for 14-18 hours at 37°C, 250rpm
- *Can use a different helper phage if needed. In step 4, add the antibiotic specific to the Helper Phagemid
Day 2
1. Spin culture at 4,000 x g for 10 minutes
2. Transfer supernatant to a fresh conical. Repeat spin on supernatant
3. Transfer the upper 90% of supernatant to a new conical
4. Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix
5. Incubate at 4°C for at least 60 minutes
6. Centrifuge at 12,000 x g for 10 minutes. Carefully decant
7. Spin again briefly
8. Gently resuspend pellet in 1.6mL 1x TBS
9. Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes
10. Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes
11. Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each
12. Let sit at room temperature for 5 minutes
13. Spin at 1300 x g for 10 minutes
14. Decant the supernatant
15. Spin briefly. Remove supernatant with pipet
16. Resuspend pellets in 300ul TE
17. Phenol extraction: add 300ul phenol. Vortex for 15 seconds
- a. Let sit for 15 minutes. Spin for 10 minutes
18. Add H2O so volume samples is about 300ul
19. Phenol/chloroform extraction*: add 300ul PCIA
- a. Vortex for 15 seconds. Spin for 10 minutes
20. Repeat Phenol/Chloroform extraction
21. Chloroform extraction*: add 300ul chloroform
- a. Vortex 15 seconds. Spin for 10 minutes
22. Add 30ul 2.5M NaAc (pH 4.8)
23. Add 2-2.5 volumes ethanol
24. Let precipitate for ~2 hours at -20°C
25. Spin for 1 minute
26. Decant supernatant
27. Resuspend in 25-50ul TE
- *Performing steps at 4°C helps with separation
2.5M NaCl/20% PEG-8000 (5x)
- PEG-800 100g
- NaCl 75g
- H2O 400mL
- *Bring final volume to 500 mL
TBS (1x)
- Tris 6.05 g
- NaCl 8.76 g
- H2O 800 mL
- *Adjust pH to 7.6 with 1M HCl
- *Adjust volume to 1 L
M13 Amplification
This protocol is to make more M13 phages.
Need
- Fresh plate of infectable cells (contain F’ episome)
Day 1
1. Grow liquid overnight culture of infectable cells
Day 2
1. Add 200ul overnight culture to 20mL LB in a 250mL flask
2. Add 1ul phage suspension
3. Incubate for 4-5 hours at 37°C, 250rpm
4. Centrifuge for 10 minutes at 4500 x g
5. Transfer supernatant to a new tube.
6. Repeat centrifugation on supernatant
7. Transfer top 16mL of supernatant to a new tube
8. Add 4mL of 2.5M NaCl/20% PEG-8000. Briefly mix
9. Precipitate phage for 1 hour or overnight at 4°C
10. Centrifuge for 15 minutes at 12000 x g. Decant supernatant
- a. Spin briefly. Remove residual supernatant with pipet
11. Resuspend pellet in 1mL 1x TBS. Transfer to 1.7mL tube
12. Spin briefly to remove cell debris
13. Transfer supernatant to a new tube
14. Add 200ul of 2.5M NaCl/20% PEG-8000
15. Incubate on ice for 15-60 minutes
16. Spin for 10 minutes at 12000-14000 rpm. Discard supernatant
17. Briefly spin. Remove supernatant with pipette
18. Resuspend pellet in 200uL TBS
19. For long term storage at -20C, add 200uL glycerol
2.5M NaCl/20% PEG-8000 (5x)
- PEG-8000 100 g
- NaCl 75 g
- H2O 400 mL
- *Bring final volume to 500 mL
TBS (1x)
- Tris 6.05 g
- NaCl 8.76 g
- H2O 800 mL
- *Adjust pH to 7.6 with 1M HCl
- *Adjust volume to 1 L
- *Store at 3C for up to 3 months
Amplification of Phage using Helper Phage
Need
- Plate of infectable cells that contain F’ episome
- 2.5M NaCl/20% PEG-8000
- 1x TBS
Day 1
- Add a fresh colony of infectable cells to 50mL LB in 125mL flask.
- a. Include phagemid antibiotic only.
- b. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
- Add the helper phage to a final concentration of 1 x10^8 phage/mL
- Incubate for 60-90 minutes, shaking
- Add Helper Phagemid antibiotic to a high concentration
- Grow for 14-18 hours at 37°C, shaking
Day 2
- Spin culture at 4,000 x g for 10 minutes
- Transfer supernatant to a fresh conical. Repeat spin on supernatant
- Transfer the upper 90% of supernatant to a new conical
- Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix
- Incubate at 4°C for at least 60 minutes
- Centrifuge at 12,000 x g for 10 minutes. Carefully decant
- Spin again briefly
- Gently resuspend pellet in 1.6mL 1x TBS
- Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes
- Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes
- Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each
- Let sit at room temperature for 5 minutes
- Spin at 1300 x g for 10 minutes
- Decant the supernatant
- Spin briefly. Remove supernatant with pipet
- Resuspend pellet in 200ul 1x TBS.
- a. If desired, combine contents of both tubes into one
2.5M NaCl/20% PEG-8000 (5x)
- PEG-8000 100 g
- NaCl 75 g
- H2O 400 mL
- *Bring final volume to 500 mL
TBS (1x)
- Tris 6.05 g
- NaCl 8.76 g
- H2O 800 mL
- *Adjust pH to 7.6 with 1M HCl
- *Adjust volume to 1 L
Bacterial Transformation Using Frozen Competent Cells
Before you start
- Heat hot plate or water bath to 42°C
- Warm selection plates to 37°C
Transformation
- Thaw chemically competent cells on ice for 10-15 minutes
- Add 40ul cells to fresh 1.7mL tube
- Add DNA
- a. If using a ligation product add up to 10ul of sample
- b. If using supercoiled plasmid add 100ng
- Incubate on ice for 30 minutes
- Heat shock cells on hot plate (or water bath) for 30-45s* @ 42°C
- Incubate on ice for 2-5 minutes
- Add 200 μL SOC and shake gently for 1-2 hours @ 37° C
- a.(Note: Can also recover in 960ul. After recovery, gently spin cells and remove supernatant. Resuspend in 200ul LB)
- Plate 100-200ul cells onto selection plates
- a. If high efficiency is expected, we suggest also plating a 1:10 dilution
- Once dry, turn upside down (agar on top) and incubate overnight @ 37° C
- *Optimal timing depends on cells
SOC (1L)
- Tryptone 20 g
- Yeast Extract 5 g
- MgSO4 4.8 g
- Dextrose 3.6 g
- NaCl 0.5 g
- KCl 0.186 g
Bacterial Conjugation
Need
- Donor cells: Cells already containing F’ episome
- Recipient cells: Cells with a resistance marker that is absent from donor cells
- Double selection plate containing antibiotic to select for F’ episome and a second antibiotic to select for the recipient cells
Day 1
- Set up liquid overnight of donor cells. Include antibiotic
- Set up liquid overnight of recipient cells. Include antibiotic
Day 2
- Mix 500ul of each overnight sample in a new tube. Mix by pipetting or flicking
- Incubate for 30 minutes at 37°C, shaking
- Plate 100ul onto double selection plate
- a.We advise also plating a 1:10 dilution
- Incubate at 37°C
http://inst.bact.wisc.edu/inst/index.php?module=Book&func=displayarticle&art_id=128
Making Chemically Competent Bacteria
To prepare for Day 2
- Set centrifuge to 4°C
- MgCl2 and CaCl2 solutions
- Thaw DMSO
- Chill tubes
- Acquire liquid nitrogen or dry ice
Day 1
- Grow cells O/N
Day 2
- Add 0.5mL of the overnight culture to 50mL LB
- Grow until OD is between 0.2 and 0.4
- Incubate on ice for 30 minutes
- Centrifuge for 10 minutes at 2700 x g and 4°C
- Decant. Dry upside down on a paper towel for 1 minute
- Completely resuspend in 30mL 0.8M MgCl2, 0.2M CaCl2
- a. Gently vortex
- Centrifuge for 10 minutes at 2700 x g and 4°C
- Decant. Dry upside down on a paper towel for 1 minute
- Fully resuspend in 2mL of 0.1M CaCl2
- Chill sample on ice. Add 70ul DMSO, keeping the sample tube on ice
- Swirl to mix
- Incubate on ice for 15 minutes
- Add 70ul DMSO, swirl to mix, keeping the sample tube on ice
- Dispense 200ul into pre-chilled 1.7mL tubes
- Snap freeze with liquid nitrogen or dry ice
- Store at -80°C until ready to use
