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From 2014.igem.org

Contents 1 Phage Amplification 2 Heat Shock Transformation 3 Bacterial Conjugation 4 CaCl2 Competent Cells 5 Phagemid DNA Isolation 6 M13 Amplification 7 Mini-prep 8 Bacterial Infection 9 Cloning a new spacer into pCas9

Phage Amplification

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

Protocol from “Use of M13KO7 Helper Phage for isolation of single-stranded phagemid DNA” by NEB

Heat Shock Transformation

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

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

CaCl2 Competent Cells

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

Phagemid DNA Isolation

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

Protocol from: “Use of M13K07 Helper Phage for isolation of single-stranded phagemid DNA” by NEB

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

Adapted from M13 Amplification protocol from NEB

Mini-prep

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

Protocol and Reagent recipes from QIAGEN unless stated otherwise

Bacterial Infection

Need

• Fresh plate of cells that contain the F’ episome

Day 1

1.Pick colony from plate and grow overnight

Day 2

1.Dilute overnight to OD600 0.1

2.Incubate until OD600 ~ 0.5*

3.Add phage to a final concentration of 1 x108 phage/mL

4.Incubate for 30 minutes to infect

5.Plate samples under selection

a.We recommend trying a range of dilutions

Note: All incubations are done at 37°C and shaking at 225rpm

Note: If multiple samples are to be done in parallel

• If using the same cells, we suggest growing one large batch of cells. Once the OD has reached ~0.5, divide into 50mL samples then add phage.
• If using different cells, grow each sample to ~0.5 then store on ice. Once all samples have reached ~0.5, incubate on ice for another 30 minutes. Warm for 20-30 minutes at 37C, 250 rpm. Measure OD again to check that samples are comparable (yes, they will have grown some). Add phage

Protocol from “Eliminating Helper phage from Phage Display

Cloning a new spacer into pCas9

Design the new spacer

1. Find a PAM site (5’- NGG -3’). This will be on the 3’ end of the targeting sequence

2. The 30bps upstream of NGG motif (on the same strand) will be the spacer

3. Oligo I (fwd. primer): To the spacer sequence, add AAAC to the 5’ end and G to the 3’ end

a. NOTE: These nucleotides are for ligation purposes and are NOT used in targeting. Therefore, they do not have to be present in the target sequence and do not count towards the 30bps

4. Oligo II (rvs. primer): Find the reverse complement of the spacer sequence. Add CAAAA to the 5’ end.

Phosphorylation

1.Mix the following reactants

Reactant	Amount
Oligo I (100uM)	1 ul
Oligo II (100uM)	1 ul
10x T4 Ligase Buffer	5 ul
T4 PNK	1 ul
H2O	42 ul

2.Incubate for 60 minutes at 37C then 20 minutes at 65C

Anneal Oligos

1. Add 2.5 ul of 1M NaCl to the phosphorylation product

2. Incubate for 5 minutes at 95C then let slowly cool to room temperature.

a. It is recommended to use a thermocycler that either cools at a gradient or steps down in temperature over the course of ~2 hours.

b. Alternatively, incubate the sample at 95C for 5 minutes then remove the heat block from the heater and let it cool naturally for 2 hours

3. Dilute the annealed oligos 1:10

Vector digest with BsaI

1. Mix the following reactants

Reactant	Amount
Cutsmart Buffer	5 ul
BsaI enzyme	1 ul
DNA	1-2ug
H2O	Bring volume to 50ul

2. Incubate for 90 minutes at 37C then 20 minutes at 65C

3. Gel extract digested pCas9

Ligation

1. Mix the following reactants

Reactant	Amount
Gel extracted vector	1 ul
Diluted annealed oligos	2 ul
10x T4 Ligase Buffer	2 ul
T4 Ligase	1 ul
H2O	14 ul

2. Incubate overnight (8-12 hours) at 16C

Transform

1. Transform into DH5alpha

Protocol from Marraffini Lab