Stock Solutions: *
Glucose minimal plates: *
PEG/NaCl: *
2x YT media: *
1x YT media: *
GROWTH OF M13 SINGLE-STRANDED DNA *
Phagemid Single strand DNA preparation. *
M13 RF (double strand) DNA PREPARATION *
Alternate M13 RF DNA Preparation *
Day 1 *
Day 2 *
Day 3 *
_________________________________________________________
M13 Phage and Phagemids
Autoclave (unless otherwise indicated) the following reagents separately and cool before mixing aseptically.
|
15 g Difco Bacto® agar in 888ml H2O |
888 ml |
|
10x M9 salts |
100 ml |
|
1 M MgSO4 |
1 ml |
|
1 M CaCl2 |
0.1 ml |
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1% thiamine HCl, sterilised by filtration |
1 ml |
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20% glucose |
10 ml |
|
------- |
|
|
Final volume |
1 l |
10x M9 Salts:
|
Na2HPO4 |
60 g |
|
KH2PO4 |
30 g |
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NH4Cl |
10 g |
|
NaCl |
5 g |
|
----- |
|
|
Final volume |
1 l |
Autoclave and store at 4oC.
|
polyethylene glycol 6000 |
20 g |
|
NaCl |
14.6 g |
|
------- |
|
|
Final volume |
100 ml |
Autoclave and then store at 4oC.
|
Bacto tryptone |
16 g |
|
|
Bacto yeast extract |
10 g |
|
|
NaCl |
5 g |
|
|
----- |
||
|
Final volume |
1 l |
Dissolve ingredients in H2O and adjust pH to 7.2 with NaOH. Autoclave the same day.
|
Bacto tryptone |
8 g |
|
Bacto yeast extract |
5 g |
|
NaCl |
5 g |
|
---- |
|
|
Final volume |
1 l |
Dissolve ingredients in H2O and adjust pH to 7.2 with NaOH. Autoclave the same day.
YT plates:
Add 15 g/litre Difco agar to YT media (3 g/200 ml). Autoclave the same day.
Soft agar:
Add 6 g/litre to YT media (1.2 g/200 ml). Autoclave the same day.
Glucose/Tris/EDTA:
|
50 mM Glucose |
4.5 g |
|
10 mM EDTA |
10.0 ml of 500 mM Na EDTA pH 8 |
|
25 mM Tris HCl pH 8.0 |
12.5 ml of 1 M Tris HCl 8.0 |
|
------ |
|
|
Final volume |
500 ml |
Autoclave at low pressure.
NaOH/SDS:
|
20% SDS |
25 ml |
|
2 M NaOH |
50 ml |
|
------ |
|
|
Final volume |
500 ml |
Leave in cold room until cloudy. Store at 4oC
GROWTH OF M13 SINGLE-STRANDED DNA
(D. R. Smith)
Day 1
1. Inoculate 10 ml YT media with a single colony of host bacteria (i.e. TG1) and incubate overnight at 37oC with shaking.
Day 2
2. Inoculate 100 ml 2x YT media with 1 ml of overnight culture of TG1.
3. Immediately dispense 1.5 ml aliquots into Sterilin universal tubes.
4. Inoculate each tube with a colourless plaque using a sterile wooden cocktail stick. (Not a toothpick as these are often treated with anti-microbial agents.)
5. Shake tubes at 37oC for 5 hours. Transfer to microcentrifuge tubes.
6. Centrifuge for 5 mins in Microcentrifuge. (This and subsequent steps can be performed at room temperature). Pour supernatant into a fresh tube, being very careful not to pick up any cells - do not take all the supernatant.
7. If you cannot process all the recombinants your require at this stage, the supernatants can be stored at 4oC for 24 hours.
Re-centrifuge before next stage.
8. Re-centrifuge supernatant as before, to ensure that all cells are removed.
9. Add 200 ul PEG/NaCl to supernatant. Shake, then leave to stand for 15 mins.
10. Centrifuge for 5 mins. Discard supernatant.
11. Centrifuge 2 mins. Carefully remove all remaining traces of PEG with a drawn out pasteur pipette. Wipe off any traces of PEG on the mouth of the tube with a tissue.
A viral pellet should be observable at this stage.
12. Add 100 ul TE buffer to the viral pellet. Resuspend well. Add 50 ul of phenol saturated with Te buffer. Vortex 15-20 seconds.
13. Stand tubes for 15 minutes at R/T. Vortex 15 seconds.
14. Centrifuge for 3 mins.
15. Remove and transfer upper (aqueous) layer to a fresh microcentrifuge tube.
16. Add 10 ul 3 M NaAc. Add 250 ul ice cold EtOH>
17. Leave overnight at -20oC to precipitate DNA.
Day 3
18. Centrifuge for 10 mins at 4oC. Wash pellet with 1 ml ice cold 80% EtOH. Pour off ethanol and leave to drain until dry.
19. Redissolve in 50 ul TE buffer. Store at -20oC.
Note 1
Some investigators have found it necessary to extract with 1 volume chloroform prior to ethanol precipitation in order to remove all traces of PEG (which will result in spurious bands in sequencing). However if stage 11 is performed carefully this step is not needed.
Note 2
Many procedures include an ether extraction step (washing twice with 500 ul of diethyl ether) after stage 15. However, the final ethanol precipitation will remove phenol to give a single stranded DNA preparation sufficiently pure for the sequencing reaction.
Phagemid Single strand DNA preparation.
Adapted from Promega protocol, T. Moss.
Transform phagemid DNA into JM109 or TG1 and grow an O/N culture from a single colony using the appropriate antibiotic selection.
(Note: It is a good idea to check by duplicate plating that the colony will grow on minimal plates (see M13 and Phagemid protocols) plus appropriate antibiotic)
Dilute O/N culture 50x into fresh LB-antibiotic, e.g. 100ul into 5ml in 50ml flask, (keep nominal flask volume between 10 and 20x culture volume).
Grow at 37oC for 30 to 40 min. then add a 10 to 20 fold excess of phage M13KO7, e.g. for a 5ml culture use about 1010 p.f.u.
Grow culture for 6 to 7 hr or O/N for about 14hr (Note: 24 hr is too long as bacteria begin to lyse and much genomic DNA is recovered.
Spin culture 10krpm, 10-15 min. (~10kxg), recover S/N and respin to remove trace bacteria.
Add 0.25 vol. of 20% PEG (MW 8,000), 3.75M Amm.Acetate, pH7.5. Mix and leave on ice 30min. Spin 10kxg, 10-15 min. and drain tube very well.
Resuspend phage pellet in 400ul TE (up to 20 ml culture), and remove excess PEG by one chloroform extraction, vortexing well for a full minute. Extract ssDNA with phenol/chloroform 3 to 4 times, until interface is clear, again vortexing very well at each step. Finally, extract once more with chloroform and precipitate at -20oC by adding Na Acetate to 0.3M and 2-3 vol. EtOH.
Expect 1-2 ug phagemid per 5ml of culture and about the same amount of helper phage.
Notes:
1) EtBr staining of s.s. phage DNA is some 5x less intense than d.s. DNA.
2) M13KO7 s.s. is 8.7kb and runs against linear double strand markers at about 3.5kbp, pT3T7u18/19 s.s. runs around the 1.5kbp position.
M13 RF (double strand) DNA PREPARATION
(modified by D.R. Smith)
Day 1
1. Inoculate 10 ml of L-broth (or YT media) with a single colony of host E. coli (i.e. TG1, JM 101 or JM109) from a minimal plate, and incubate at 37oC with shaking overnight (~300 rpm).
Day 2
2. Inoculate 10 ml 2x YT media with 0.5 ml of overnight culture of TG1. Label tubes "competent cells". Incubate at 37oC with shaking for 3 hours. (This will provide enough cells for 3 x 200 ml preps, so multiply accordingly.)
3. Inoculate 10 ml of 2x YT media with single colony of TG1 from minimal plate (this will provide enough fresh cells for ten 100 ml cultures). Label tubes "fresh cells". Incubate at 37oC with shaking for 3 hours.
4. Spin down cells grown for competent cells in Sorvall RC-3B at 4,000 rpm, 20 mins and 4oC. Resuspend in 1/2 volume 50 mM Cacl2 (ice cold). Incubate on ice for 20 mins. After 20 mins re-centrifuge cells (4,000 rpm, 10 mins, 4oC) and resuspend in 1/10 th volume 50 mM CaCl2. Incubate on ice until required. These competent cells will keep on ice for some hours. After 24 hours the efficiency of transformation begins to decrease, although cells may still be usable 4-5 days later.
5. Inoculate 100 ml 2x YT media with 1 ml of the fresh grown culture. Incubate at 37oC with shaking for about 2 hours.
6. To 300 ul competent cells add 0.6 ul of ssM13 DNA (from sequencing solution i.e. ~50 ng/ul). Incubate for 40 mins on ice. Heat shock at 42oC for 2 mins. Return to ice. Add transformed competent cells to growing 100 ml cultures. Incubate overnight at 37oC with shaking.
Day 3
7. Spin down cultures in Sorvall RC-3B at 4,000 rpm, 20 mins and 4oC. If possible spin down in 50 ml Oakridge tubes (x3 spins, then there is no need to transfer cells).
8. Resuspend in 3 ml of 50 mM glucose, 10 mM EDTA, 25 mM Tris HCl pH 8.
9. Add 6 ml of ice cold 0.2 M NaOH, 1% SDS, mix very vigorously and leave on ice for about 15 mins with occasional vigorous shaking.
10. Add 3 ml ice cold 3 M KHAc pH 4.8. Mix with vigorous shaking and leave on ice for 20 mins with occasional vigorous shaking.
11. Spin in Sorvall RC-5B, HB4 rotor at 10,000 rpm, for 15 mins and 4oC. Then filter supernatant through sterile gauze and transfer to fresh Oakridge tube.
12. Precipitate nucleic acid by the addition of 7.5 ml isopropanol (0.6 vols) and centrifuge immediately at 10,000 rpm in Sorvall, setting temperature to 22oC.
13. Pour off supernatant and discard. Dissolve pellet in 1 ml of 100 mM Tris HCl pH 8.3, 10 mM EDTA (10x TE) and transfer to 4 ml tube. (Note that it is often useful to run a quick agarose gel of the dissolved sample at this stage, two samples of 1 ul and 10 ul being about optimal. The gel can be run while samples are undergoing the next step. This often saves much time if the prep has not worked).
14. Add 50 ul of RNase A (20 mg/ml) and incubate at 37oC for 30 mins. Add 100 ul of 3 M NaCl (or NaAc).
15. Add 1 ml of phenol (0.1% hydroxyquinoline) saturated with 0.3 M TNE, vortex well and centrifuge for 10 mins at 10,000 rpm in Sorvall RC-5B.
16. Remove upper phase into a fresh 4 ml tube and repeat (15) once or twice until no interface is seen.
17. Add 1 ml of chloroform, vortex and spin as above. Remove upper phase into fresh 4 ml tube.
18. Precipitate nucleic acid with 2 ml of ice cold EtOH, mix well and incubate at -20oC for 30 mins.
19. Spin tube at 10,000 rpm for 15 mins and 4oC in Sorvall RC-5B, HB4 rotor. Pour off supernatant and discard, leaving the tube to drain for a minute.
20. To remove bulk of RNA by differential precipitation, redissolve pellet in 1 ml of 0.3 M NaAc pH 7 at room temp. Add 570 ul R/T isopropanol dropwise on a whirlimix. Leave 10 mins at R/T then centrifuge for 15 mins at 10,000 rpm in Sorvall RC-5B, HB4 rotor at R/T. Pour off supernatant (but retain in case M13 not precipitated). Wash pellet with 1 ml R/T 0.3 M NaAc/isopropanol (1:0.6) and spin for 5 mins at R/T 10,000 rpm.
21. Discard supernatant and wash with 1 ml of ice cold EtOH spin as above setting temperature to 4oC.
22. Discard supernatant and leave tube to drain for a few minutes. When DNA dry, re-dissolve in 100 ul of 1x TE. About 100 ug RF M13 can be obtained from a 100 ml culture.
Alternate M13 RF DNA Preparation
(R.F. de Winter).
1. Inoculate O/N culture of the appropriate host (e.g. TG1) in 1x YT medium (L-broth). Shake at 37oC.
2. Dilute O/N culture 1:50 in YT medium. Grow until OD 660 nm (1 cm) = 0.6. Mix 1.25 ml culture with 40 ul high titer phage supernatant or white plaque. Shake at 37oC for 2.5 hr. Shake vigorously (300 rpm at shaker).
Leave O/N culture out on bench.
3. After 2.5 hr add 1 ml of O/N culture to phage culture. Vortex well (for aeration). Shake at 37oC for 2.5 hr. This mixture is the phage stock.
4. Add phage stock to 500 ml of pre-grown uninfected culture in YT medium which is prepared as follows:- 3.25-3.5 hr before final inoculation 1 ml of the O/N culture is diluted in 50 ml YT. Shake at 37oC for 2.5 hr. Then add 10 ml of this culture to 500 ml of YT medium. Shake for 1 hr at 37oC before adding the pre-grown phage stock. Grow this mixture overnight at 37oC.
5. Isolate RF DNA either using the Birnboim and Doly method or the Cleared Lysate method. The supernatant can be used for large scale isolation of single stranded DNA.