Team:CU-Boulder/Notebook/Protocols
From 2014.igem.org
Contents |
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)
- 100 g PEG-800
- 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 1L
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)
- 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
- Store at 3C for up to 3 months
QIAprep Spin Miniprep (Centrifuge method)
- This protocol is taken from the Qiagen Mini-prep Kit and is used to isolate plasmid DNA from a bacterial overnight.
Notes before starting
- Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000
- Add the provided RNase A solution to Buffer P1, mix, store bottle at 2-8°C
- Add ethanol (96-100%) to Buffer PE before use
- All centrifugation steps are carried out at 13,000 rpm (~17,900xg) in a conventional table-top microcentrifuge
- 1.Centrifuge 1-6mL bacterial overnight culture at >8000 rpm (6800xg) for 3 minutes at room temperature (15-25C)
- 2.Resuspend pellet in 250ul Buffer P1 and transfer to microcentrifuge tube
- 3.Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear.
- a.DO NOT allow lysis reaction to proceed for more than 5 minutes
- b.If using LyseBlue reagent, the solution will turn blue
- 4.Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
- a.If using LyseBlue reagent, the solution will turn colorless
- 5.Centrifuge for 10 minutes
- 6.Apply supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through
- 7.Recommended: Wash the QIAprep spin column by adding 500 ul Buffer PB. Centrifuge for 30-60 s and discard the flow-through
- a.Only required when using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content
- 8.Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 30-60 s and discard the flow-through
- 9.Centrifuge for 1 minutes to remove residual wash buffer
- 10.Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 30ul Buffer EB. Let stand for 1 min, and centrifuge for 1 minute
- a.Can elute DNA in 50ul but this will decrease DNA concentration
- b.To increase yield, let sit for up to 4 minutes
Buffer Recipes
P1 50 mM Tris-Cl, pH 8.0 10 mM EDTA 100 ug/mL RNase A
- *After RNase A addition, the buffer should be stored at 2-8C
P2 (Lysis buffer)
- 200 mM NaOH
- 1% SDS (w/v)
N3*
- 4.2 M Gu-HCl
- 0.9 M KAc, pH 4.8
PB*
- 5 M Gu-HCl
- 30% isopropanol
PE*
- 10 mM Tris-HCl pH 7.5
- 80% ethanol
Elution Buffer (EB)
- 10 mM Tris-CL, pH 8.5
- Recipes from OpenWetWare