Archives: Methods

Objective:

To isolate phage resistant mutants

Reagents:

• Overlay agar in tubes: 0.6% agar, 1% PP2
• 1% PP2 plates.
• Host bacerial culture

Methods:

Overlay Method

1. Grow host bacteria overnight. Dilute 1:10.
2. Add 0.1 ml phage stock solution and 0.1 ml of diluted bacterial culture into prewarmed overlay agar (temperature maintained at 45ºC).
3. Swirl overlay agar and pour onto a PP2 plate. Do at least 3 plates to increase your chances of getting mutants.
4. Incubate at 37ºC. for 2-3 days.
5. Pick individual colonies with sterile toothpicks onto fresh PP2 plates. Streak for single colony. Incubate at 37ºC. overnight.
6. Freeze mutants with DMSO.

Spreading Method

1. Add 0.1 ml phage solution (10^-2 dilution) and 0.1 ml diluted bacterial culture onto a PP2 plate and spread plate.

NOTE: This method does not give uniform lawns as predictably as the overlay method, but you don’t have to make the overlay agar.

1. Dilute an overnight culture of host cells (DH5a, JM101, NM522) 1:100 in 25 ml TB broth and infect with a single plaque of recombinant M13.
2. Shake vigorously for 6 hours at 37ºC.
3. Collect the supernatant by centrifugation at 12,000 x g for 15 minutes. Pour supernatant into a fresh tube and repeat.
4. Precipitate the phage by the addition of 0.25 volumes of phage precipitation solution and incubate on ice for 30 minutes, then centrifuge at 12,000 x g for 15 minutes. Thoroughly drain supernatant and wipe excess away with a Kimwipe.
5. Resuspend pellet in 400 ul TE.
6. Add 1 volume of 24:1 cholorform:isoamyl alcohol and vortex for 1 full minute. Spin at 12,000 x g for 5 minutes.
7. Transfer the aqueous phase to another tube; leave the interface behind. Add 1 volume of TE saturated phenol/chloroform, vortex for 1 minute, centrifuge at 12,000 x g for 5 minutes.
8. Repeat above step until there is no longer an interface present.
9. Transfer the aqueous phase to a fresh tube and add 0.5 volumes 7.5 M ammonium acetate, pH 7.5 and 2 volumes of 100% ethanol. Mix and incubate at -20 for 30-90 minutes.
10. Centrifuge at 12,000 x g at 4ºC for 15 minutes, rinse the pellet with 70% ethanol and dry at bench for 3-5 minutes.
11. Resuspend the pellet in 50 µl of deionized water.

Phage precipition solution:

• 3.75 M ammonium acetate
• 20% PEG-8000
• Add equal volumes of 40% PEG-8000 and 7% M NH4OAc, pH 7.5
• TE-saturated phenol/chloroform
• Mix equal parts of TE and phenol and allow the phases to separate. Then mix 1 part of the lower, phenol phase with 1 part chloroform:isoamyl alcohol (24:1).

References:

• TnphoA – Manoil and Beckwith. PNAS. 82: 8129-8133. 1985;
• lambda-TnphoA – Gutierrez et. al.. J. Mol. Biol. 195: 289-287. 1987.

Method:

1. Before starting the mutagenesis protocol, ensure the plasmid you wish to mutagenize is in an appropriate E. coli background such as TB1 (C268 in our culture collection). Otherwise, you will only succeed in propagating phage!
2. Grow up your strain carrying your plasmid of interest in 5 ml of LB supplemented with 10 mM MgSO₄ and appropriate antibiotic selection to an O.D.₆₀₀ of 0.5 (approximately 2 x 10⁸ cells/ml).
3. To 1 ml of cells (2 x 10⁸ cells) add 2 x 10⁸ lambda-TnphoA phage particles (multiplicity of infection of 1).
4. Incubate at 30ºC for 15 minutes on a tube roller.
5. Set up 10 tubes containing 100 µl of infected culture with 1 ml of LB and antibiotic selection for the plasmid of interest (i.e. no kanamycin selection yet).
6. Incubate the tubes at 30ºC for 4 hours on a tube roller.
7. Dilute 50 µl of culture into 5 ml of LB with selection for plasmid and 50 µg/ml kanamycin (selection for transposon). Grow overnight at 30ºC.
8. Prepare a separate plasmid preparation from each sample using the alkaline lysis miniprep method. (Protocol in this manual.)
9. Use these plasmid preparations to transform E. coli CC118 (C494 in our culture collection). This strain lacks funtional alkaline phosphatase.
10. Plate transformants on LB containing selection for the plasmid, kanamycin and 20 µg/ml 5-bromo-4-chloro-3-indolyl phosphate (XP). XP is a substrate for alkaline phosphatase. Double resistant transformants represent successful transpositions onto plasmid DNA; blue double resistants are producing functional alkaline phosphatase.

Notes On Protocol:

Avoid using ampicillin for selection as complications may arise from synergistic and antagonistic effects of double selecting with ampicillin and kanamycin later on in the protocol. Otherwise, use 100 µg/ml ampicillin rather than 50 if it is necessary to use ampicillin selection. Stock solution of XP is prepared by dissolving the p-toluidine salt of XP in DMSO; store at -20ºC. XP is light sensitive so stocks and plates should be treated accordingly. Frequency of blue transformants is approximately 1 to 5%. Phage lambda-TnphoA is propagated on C493; working stocks of phage can be stored at 4ºC but reserve stock solutions should be stored at -70ºC. When performing colony immunoblots or Western blots of TnphoA mutants, use the horseradish peroxidase development system rather than the alkaline phosphatase system.

Objective:

To isolate phage resistant mutants.

Reagents:

• Overlay agar in tubes: 0.6% agar, 1% PP2
• 1% PP2 plates.
• Host bacerial culture

Methods:

Overlay Method

1. Grow host bacteria overnight. Dilute 1:10.
2. Add 0.1 ml phage stock solution and 0.1 ml of diluted bacterial culture into prewarmed overlay agar (temperature maintained at 45^oC).
3. Swirl overlay agar and pour onto a PP2 plate. Do at least 3 plates to increase your chances of getting mutants.
4. Incubate at 37^oC. for 2-3 days.
5. Pick individual colonies with sterile toothpicks onto fresh PP2 plates. Streak for single colony. Incubate at 37C. overnight.
6. Freeze mutants with DMSO.

Spreading Method

1. Add 0.1 ml phage solution (10^-2 dilution) and 0.1 ml diluted bacterial culture onto a PP2 plate and spread plate.

Note: This method does not give uniform lawns as predictably as the overlay method, but you don’t have to make the overlay agar.

1. Grow cells in 2-5 ml broth to late log phase.
2. Pellet 1-2 ml cells in microfuge.
3. Resuspend cells in 400 ul TES (50 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, pH7.5).
4. Add: 17 ul 30% (w/v) sarkosyl (final concentration of 1%) and 5 ul 10 mg/ml proteinase K (final concentration 100 ug/ml).
5. Incubate at 37C for 30-60 minutes, or until the solution clears.
6. Add 400 ul 4 M NH4OAc and mix well.
7. Extract 2X with 1:1 phenol:chloroform.
8. Extract 1X with chloroform.
9. Precipitate with an equal volume of isopropanol at RT for 10 minutes.
10. Resuspend pellet in 400 ul 0.1 M NaOAc pH 6.0. Pellet may not resuspend completely. Reprecipitate DNA with 800 ul EtOH for 15 minutes at RT.
11. Centrifuge at 11K for 15 minutes to pellet DNA.
12. Rinse pellet in 1 ml 70% EtOH for 5 minutes.
13. Dry pellet by placing open tube in 37C incubator or on bench top for 15-20 minutes.
14. Resuspend DNA in 50 ul TE (10 mM Tris pH 8.0, 0.1 mM EDTA) overnight at 4C. May add 1 ul RNase to help DNA dissolve. Yields 25-50 ug of DNA; use 1-5 ul per digest.

TES:

• 5.0 ml 1 M Tris pH 7.5
• 0.2 ml 5 M NaCl
• 2.0 ml 0.5 M EDTA
• 93 ml sterile MQ water

Materials:

The DEAE paper can be used as supplied by Schleicher and Schuell, however the binding capacity can be increased by washing the paper in 10mM EDTA, pH 7.5 for 10 minutes, 0.5M NaOH for 5 minutes, followed by several rinses in distilled water. The paper can be stored at 4^oC for several months.

NET buffer:

• 0.15M NaCl
• 0.1mM EDTA
• 20mM Tris-HCl, pH 8.0

High salt NET buffer:

• 1.0M NaCl
• 0.1mM EDTA
• 20mM Tris-HCl, pH 8.0

Method:

1. After the DNA bands have been separated electrophoretically (at around 80 V), the gel is viewed under long wave UV light and a slit cut in the gel, just ahead of the desired band. A piece of DEAE paper is inserted in the slit. Another piece of DEAE paper can be inserted behind the desired band, to prevent unwanted bands being trapped by the paper.
2. Electrophoresis is continued for approximately 10 to 15 minutes with the voltage increased to 100-120 V.
3. The DEAE paper is removed and binding of the DNA checked by viewing the paper with long wave UV light. The paper is then placed in an eppendorf tube containing 500 µl NET buffer to wash away remaining agarose. The bound DNA can now be stored at 4^oC for several days. The DNA should always remain covered with buffer to prevent irreversible binding which may occur if the paper is allowed to dry out.
4. To elute the bound DNA, place the DEAE paper in an eppendorf tube containing 250 µl of high salt NET buffer so that the paper is completely covered. Make sure that the side of the paper containing the DNA faces the buffer and not the side of the tube. If necessary, centrifuge briefly to submerge the paper.
5. Incubate the tube at 55-68^oC for 45 minutes with occassional swirling (or until all the DNA has been eluted as checked under long wave UV light).
6. Remove the buffer to a fresh tube and add 50 µl high salt NET buffer to wash the membrane. Remove the additional buffer and add to the fresh tube.
7. Centrifuge the buffer for 30 seconds to pellet any remaining DEAE paper fibres and remove the buffer to a fresh tube (leaving approx. 20 µl in the bottom of the tube).
8. Extract the residual ethidium bromide with 3 volumes of water saturated butanol.
9. Remove and retain the bottom layer and add 1/20 volume of 3.0M sodium acetate, pH 5.25 and 2.5 volumes of cold absolute ethanol. Incubate at -70^oC for at least one hour. Ethanol precipitate and vacuum dry.

1. Isolate chromosomal DNA by using the CTAB protocols in current Pr M R.
2. Make a CsCl preparation of pLAFr3 (or other appropriate cosmid).
3. Cut pLAFr with BamHI phenol ext. + ETOH preparation.
4. Partially digest a sample of chromosomal DNA as per Maniatis protocol (pp. 9.24 – 9.28) in order to maximize production of 20 kb plasmid.
5. Scale up the conditions (all of the conditions!!) to obtain 200 – 500 µg of cut DNA.
6. Prepare a 10 – 40% sucrose gradient as per Maniatis (pp. 2.85 – 2.87).
7. Pool fractions containing 20 kb fragments and, after adding 3 vol. of sterile dH₂O, prep. the DNA.
8. Set up ligations (Maniatis p. 3.29) to maximize the formation of concatomers.
9. Package via “Packagene” protocol. Plate the infected bacteria on LB Tet. plates. Remember: if you are using pLAFr, you are looking for colonies, NOT PLAQUES, since it is a cosmid.
10. Check a randomly selected group of transformants for the presence of inserts by restriction analysis.

1. Grow cells in 2-5 ml broth to late log phase.
2. Pellet 1-2 ml cells in microfuge.
3. Resuspend cells in 400 µl TES (50 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, pH7.5).
4. Add: 17 µl 30% (w/v) sarkosyl (final concentration of 1%) and 5 µl 10 mg/ml proteinase K (final concentration 100 µg/ml).
5. Incubate at 37ºC for 30-60 minutes, or until the solution clears.
6. Add 400 µl 4 M NH4OAc and mix well.
7. Extract 2X with 1:1 phenol:chloroform.
8. Extract 1X with chloroform.
9. Precipitate with an equal volume of isopropanol at RT for 10 minutes.
10. Resuspend pellet in 400 µl 0.1 M NaOAc pH 6.0. Pellet may not resuspend completely. Reprecipitate DNA with 800 µl EtOH for 15 minutes at RT.
11. Centrifuge at 11K for 15 minutes to pellet DNA.
12. Rinse pellet in 1 ml 70% EtOH for 5 minutes.
13. Dry pellet by placing open tube in 37C incubator or on bench top for 15-20 minutes.
14. Resuspend DNA in 50 ul TE (10 mM Tris pH 8.0, 0.1 mM EDTA) overnight at 4C. May add 1 µl RNase to help DNA dissolve. Yields 25-50 ug of DNA; use 1-5 µl per digest.

TES:

• 5.0 ml 1 M Tris pH 7.5
• 0.2 ml 5 M NaCl
• 2.0 ml 0.5 M EDTA
• 93 ml sterile MQ water

Reference:

GATA 1993; 10(5):113-115.

Method:

1. End repair: Add 5-10 units of T4 DNApol and incubate at 37ºC for 5 minutes.
2. Make solution to 2.5 M NH4OAc and add 1 volume of 95% ethanol; let sit for 5 minutes at RT and spin at 14,000 x g for 20 minutes.
3. Wash with 70 % ethanol, dry and resuspend in 20 ul T4 polynucleotide kinase, containing 20 pmol ATP and 5-10 units T4 polynucleotide kinase. Incubate at 37ºC for 1 hour.
4. Ligation reaction: Blunt end ligation is carried out in the presence of 5% PEG 8000. A microgram of Bluescript SK+ is blunt ended (cut with EcoRV) and ligated with 10-100 ng of the purified PCR product in a 50 ul reaction mixture containing 1 unit of T4 ligase. Do ligation overnight using a temperature gradient of 25-2ºC.