"

From 2014.igem.org

Contents 1 Amplification of Phage using Helper Phage 2 Bacterial Transformation Using Frozen Competent Cells 3 Bacterial Conjugation 4 Making Chemically Competent Bacteria 5 Isolation of single-stranded phagemid DNA using M13K07 Helper Phage 6 M13 Amplification 7 Amplification of Phage using Helper Phage 8 Bacterial Transformation Using Frozen Competent Cells 9 Bacterial Conjugation 10 Making Chemically Competent Bacteria 11 Isolation of single-stranded phagemid DNA using M13K07 Helper Phage 12 M13 Amplification 13 QIAprep Spin Miniprep (Centrifuge method)

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

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 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

1. Set up liquid overnight of donor cells. Include antibiotic
2. 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

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)

• 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

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

1. Add the helper phage to a final concentration of 1 x10^8 phage/mL
2. Incubate for 60-90 minutes, shaking
3. Add Helper Phagemid antibiotic to a high concentration
4. Grow for 14-18 hours at 37°C, shaking

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 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

• 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)

• 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

1. Set up liquid overnight of donor cells. Include antibiotic
2. 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

1. 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

1. Set centrifuge to 4°C
2. MgCl2 and CaCl2 solutions
3. Thaw DMSO
4. Chill tubes
5. Acquire liquid nitrogen or dry ice

Day 1

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

1. Centrifuge for 10 minutes at 2700 x g and 4°C
2. Decant. Dry upside down on a paper towel for 1 minute
3. Fully resuspend in 2mL of 0.1M CaCl2
4. Chill sample on ice. Add 70ul DMSO, keeping the sample tube on ice
5. Swirl to mix
6. Incubate on ice for 15 minutes
7. Add 70ul DMSO, swirl to mix, keeping the sample tube on ice
8. Dispense 200ul into pre-chilled 1.7mL tubes
9. Snap freeze with liquid nitrogen or dry ice
10. 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 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)

• 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