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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 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

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