Bacterial Growth & plasmid preparation *

___________________________________________________________________________

Bacterial Growth & plasmid preparation

STORAGE OF E. COLI STRAINS

Note. For plasmids 1 to 10 ug of DNA should be stored at -20oC or -80oC in TE for ultimate safety.

Strains can be stored as colonies on normal L-broth plates for about 2 months at 4oC.

"Slopes"

For longer storage at 4oC and as a practical source of strain, bacteria are grown up as a lawn on "slopes", ~10 ml tubes in which 2 ml (antibiotic) L-broth agar is allowed to set while tube laying nearly flat. Produces a large surface culture.

"Stabs"

For longer storage and dispatch "stabs" are used. Small glass vials half filled with 0.5 to 1 ml (antibiotic) L-broth agar. Bacteria are stabbed deep into the agar and allowed to grow under ideal conditions 0/N, then stored at 4oC. Viable for up to 1 year.

Glycerol Cultures

To 0.5 to 1 ml of a log phase or saturated bacterial culture add ~1.2 volumes of sterile 80% glycerol in H20 (autoclaved). Store at -20oC. If stored at 70oC culture will freeze and should be thawed only once. Viable for about a year, possibly longer if freezing and thawing does not take place.

HMFM Cultures

Hogness modified freezing medium is used for storage of cultures at -70oC. Cultures are less affected by freezing and thawing in this medium (10 cycles typically gives a ten fold loss) and are viable for many years. The cultures can be sent at R/T through mail and even refrozen on arrival.

A log phase culture (OD 660 nm (1 cm) = 0.8) or even a saturated culture is centrifuged to harvest cells. After pouring off the medium, cells are resuspended in sterile:

3.6 mM K2HPO4

1.3 mM KH2PO4

2.0 mM Na3citrate

1.0 mM MgSO4.7H2O

4.4% (v/v) glycerol

and kept at -70oC. NOTE do not store at -20oC even for a short period.

Glycerol L-Broth Plates

For storage of many bacterial colonies, eg a gene bank, the colonies plated on itrocellulose are transferred to an (antibiotic) L-broth agar plate containing 20-30% glycerol (added before autoclaving agar). Plates are then sealed and stored at -20oC or -70oC. If a colony is required, filter ONLY is transferred to (antibiotic) L-broth agar plate at R/T and glycerol plate immediately replaced at -20oC. After work with filter is complete it is returned rapidly to glycerol plate in freezer.

GENERAL BACTERIAL GROWTH

L-Broth

Tryptone (Difco)	10 g		50 g
Yeast Extract (BBL or Difco)	5 g		25 g
NaCl	5 g		25 g
	-----		-----
total volume	1 litre		5 litres

Dissolve ingredients in H2O and adjust pH to 7.2 with NaOH (about 2 ml 5 M NaOH/litre, but depends on tryptone and yeast extract lots). Autoclave same day.

L-Broth Plates

12.5 gm of agar (BBL or Difco) added to 1 litre fresh L-broth and autoclaved. The agar can then be stored (mix well after autoclaving) or used immediately. Agar broth, either remelted at 100oC in microwave or freshly autoclaved, is cooled to ~65oC, antibiotic added if required and poured rapidly to a depth of ~1 cm in petri dishes. 1 litre will give about 30 x 9 cm plates.

2x YT media:

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.

1x YT media:

YT plates:

Add 15 g/litre (3 g/200 ml) Difco agar to YT media. Autoclave the same day.

YT Soft agar:

Add 6 g/litre to YT media (1.2 g/200 ml). Autoclave the same day.

Glucose minimal plates:

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
1% thiamine HCl, sterilised by filtration	1 ml
20% glucose	10 ml
	-------
Final volume	1 l

10x M9 Salts:

Na2HPO4	60 g
KH2PO4	30 g
NH4Cl	10 g
NaCl	5 g
	-----
Final volume	1 l

Autoclave and store at 4oC.

Cultures

Large cultures are grown in smooth walled flasks containing up to 20 to 25% total flask-volume of L-broth. Normally flasks are incubated at 37oC and shaken at about 220 to 250 rpm.

STOCK SOLUTIONS FOR PLASMID PREPARATIONS

Antibiotics:

For cleared lysate preparations:

50 mM Tris HCl pH 8.0

500 mM EDTA ~pH 8.3

10% Triton X-100 in H2O

3 M NaAc ~pH 7.0

Isopropanol

EtOH at -20oC

80% EtOH in H2O at -20oC

1x TE (20 mM Tris HC1, 1 mM EDTA, pH 8.3, from 100 x stock)

Phenol, 0.1% 8-hydroxyquinoline, saturated with 0.3 M TNE (stored at 4oC)

Chloroform - isoamylalcohol (24:1 v/v) (stored at 4oC)

RNAase A, 20 mg/ml in 150 mM NaAc pH 5 (made up from Sigma best grade, boiled 5 mins and then frozen in 0.5 ml aliquots -20oC)

0.3 M NaAc (from 3 M stock)

0.3 M NaAc/isopropanol (1:0.6)

 

For NaOH/SDS preparations:

50 mM glucose, 10 mM EDTA, 25 mM Tris HCl pH 8.0

0.2 M NaOH, 1% SDS

3 M KHAc pH 4.8 (KHAc dissolved initially at 6M, then titrated to pH 4.8 with glacial acetic and finally volume adjusted to give 3M K)

RNAase A 20 mg/ml (as above)

10x TE (from 100 x stock)

Phenol (as above)

EtOH at -20oC

80% EtOH in H2O at -20oC

0.3 M NaAc (from 3 M stock)

0.3 M NaAc/isopropanol (1:0.6)

 

LARGE SCALE PLASMID CULTURE-CLEARED LYSATE PREPARATION

1. Streak from a glycerol or HMFM culture onto antibiotic L-broth plates.

 

pCRI		Kanamycin	100 ug/ml
pBR322 etc	either	Tetracycline	12.5 ug/ml
	or	Ampicillin	50 ug/ml

SMALL SCALE PLASMID CULTURE-CLEARED LYSATE PREPARATION

1. Streak from a glycerol or HMFM culture onto antibiotic-L-broth plates.

 

pCRI		Kanamycin	100 ug/ml
pBR322 etc	either	Tetracycline	12.5 ug/ml
	or	Ampicillin	50 ug/ml

2. Leave to grow overnight at 37oC.

3. Transfer an isolated colony to 2 ml antibiotic-L-broth. Leave to grow for at least 6 hours, preferably until saturated, at 37oC in shaking incubator. Use sterilin universals.

4. If necessary use 1 ml of this culture to make another glycerol or HMFM culture:

Add rather more than an equal volume of autoclaved 80% aqueous glycerol (i.e. to a final concentration of 50-60%) to 1 ml of the culture. Store at -20oC.

5a. Inoculate up to 100 ml of antibiotic-L-broth with the rest of the culture (1 ml) and grow at 37oC until an OD 600 nm (1 cm) = 1.0-1.5 is reached. Add chloramphenicol to a final concentration of 170 ug/ml and shake well for at least a further 12 hours at 37oC. (N.B. The chloramphenicol stock solution is made up in ethanol - DO NOT FLAME).

or

5b. Inoculate as in 5a. and grow culture to saturation with good aeration (more than 24 hours).

6. Harvest cells in 50 ml Oakridge tubes by spinning for 20 minutes at 4,000 rpm on Sorvall RC-3B. Carefully pour off supernatant and leave tubes upturned on paper to drain for a few minutes.

7. Take up pellet in 0.5 ml of 50 mM Tris HC1 pH 8.

8. Transfer to Eppendorf tube.

9. Add 50 ul of lysozyme - 10 mg/ml, freshly made up in 50 mM tris HC1 pH 8 (final concentration 1 mg/ml). Whirlimix for a couple of seconds and leave for 5 minutes on ice.

10. Add 50 ul of 0.5 M EDTA and mix well. Leave for 5 minutes on ice.

11. Add 5 ul of RNAase A (20 mg/ml)-final concentration 100 ug/ml.

12. Add 10 ul 10% Triton and mix by gently rolling tube. Leave on ice for 15 to 20 minutes. Should be very viscous at this stage.

13. Spin in the Sorvall RC-5B, SS34 rotor for 1 to 2 hours at 18,000 rpm and 4oC.

14. Pour off the supernatant quickly but smoothly into a clean eppendorf. The supernatant should be clear and NOT viscous. Any delay in removing the supernatant will allow the viscous pellet to resuspend.

15. Add 50 ul of 3 M NaAc and an equal volume of phenol to the supernatant. Mix thoroughly by shaking or whirlimixing.

16. Spin in eppendorf centrifuge for 5 to 10 minutes and remove upper phase into fresh tube.

17. Repeat extraction with phenol once or twice until no interphase forms, then extract once with chloroform:isoamyl alcohol (24:1 v/v).

18. At this stage a small sample can be removed for a gel.

19. Precipitate plasmid and RNA with 2 vols cold ethanol and leave at -20oC for about 30 minutes.

20. Spin in Sorvall RC-5B, HB4 rotor at 10,000 rpm for 15 min, 4oC. Remove supernatant and vacuum dry pellet for 5 minutes.

21. Dissolve at room temperature in 0.5 ml, 0.3 M NaAc pH 7-8 and then differentially precipitate plasmid by adding 0.56 to 0.58 volumes (0.28 to 0.29 ml) of R/T isopropanol dropwise on whirlimixer. Leave for 10 minutes at room temperature.

22. Spin in eppendorf centrifuge for 10 minutes at R/T.

23. Wash once with 0.5 ml 0.3 M NaAc/isopropanol (1:0.6 v/v), spin 5 min R/T in eppendorf centrifuge.

24. Wash once with ice cold 80% ethanol, same spin.

25. Vacuum dry and redissolve in about 50 ul of 1x TE. Yield: can expect ~20 ug from a 50 ml culture.

 

SMALL SCALE PLASMID (COSMID) PREPARATION

(Essentially as Birnboim and Doly (1979) NAR 7, 1513-1523)

1. Grow up to a 2 to 100 ml chloramphenicol amplified culture or 2 to 25 ml saturated culture as for cleared lysate preparation. 10 ml saturated cultures can be conveniently grown in Sterilin universal tubes and yield 20ug or more of plasmid DNA.

2. 2 ml cultures are harvested directly in eppendorf tubes by repeated microcentrifugation. 10 ml cultures are harvested directly in culture tubes at 3,000 rpm and 4oC for 10 minutes in the Sorvall RC-3B using blue adaptors (conical bottomed). Larger cultures may be harvested in 50 ml or 250 ml tubes spinning for 15 minutes at 4000 rpm.

3. Resuspend cells in 200 ul of:

50 mM glucose

10 mM EDTA

25 mM Tris HC1 pH 8.0

Or simply 200 ul of TE and transfer to eppendorf tube on ice.

4. Add 400 ul ice cold 0.2 M NaOH, 1% SDS, mix and leave on ice for 5 minutes exactly.

5. Add 200 ul ice cold 3 M KHAc pH 4.8 (3 M KHAc dissolved initially at 6 M and then titrated with glacial acetic to pH 4.8, finally volume adjusted to give 3 M K).

5 ul of RNAase A (20 mg/ml) may be added at this step, but is more effective at step 8 below. Mix and leave on ice for 15 minutes.

6. Spin in eppendorf centrifuge in cold room for 5 minutes and remove supernatant into a fresh tube.

7. Precipitate nucleic acid by the addition of 500 ul of isopropanol (~0.6 volumes) and centrifuge immediately in eppendorf centrifuge for 5 minutes.

8. Remove supernatant and dissolve pellet in 300 ul 100 mM Tris HC1, 10 mM EDTA pH 8.3 (10x TE). 5 ul of RNAase A (20 mg/ml) may be added at this step and the solution incubated at 37oC for 10 to 30 min.

9. Add 300 ul phenol (0.1% hydroxyquinoline) saturated with 0.3 M TNE, whirlimix and spin in eppendorf centrifuge for 5 minutes.

10. Remove upper phase into a fresh tube. Depending on intended use of prep. step (9) can be repeated once or twice, until no interphase is seen.

11. Precipitate nucleic acid with 600 ul ice cold EtOH, mix well and leave at 20oC for more than 10 minutes.

12. Spin in eppendorf centrifuge in cold room for 10 minutes, pour off supernatant and allow tube to drain inverted.

At this stage the DNA is useable for restriction and nick translation.

If it is an advantage to remove the excess RNA the following steps may be used:-

13. Redissolve pellet in 500 ul 0.3 M NaAc ~pH 7 at R/T. Add 0.28 to 0.29 ml R/T isopropanol dropwise on whirlimix. Leave 10 minutes at R/T then spin for 10 minutes in eppendorf centrifuge at R/T.

14. Wash with 1 ml of R/T 0.3 M NaAc/isopropanol (1:0.6 v/v) and spin for 5 minutes as in (13).

15. Discard supernatant, add 500 ul chilled EtOH and spin again as in (14).

16. Discard supernatant and allow tube to drain.

Plasmid DNA should be restrictable by most enzymes and may be nick translated. 5’ labelling may also be carried out but since a small amount of low molecular weight RNA is present this will take up most of the label.

 

LARGE SCALE PLASMID (COSMID) PREPARATION

(Essentially as Birnboim and Doly (1979) NAR 7, 1513-1523)

1. Grow 500 or 1 litre culture as for cleared lysate preparation, i.e. either using chloramphenicol amplification or growth to saturation.

2. Harvest cells by spinning for 15-20 minutes at 4 krpm and 4oC in Sorvall RC-3B, (soft stop to avoid resuspension of bacterial pellet).

3. Resuspend cells in 30 ml of:

50 mM glucose

10 mM EDTA

25 mM Tris HC1 pH 8.0

and transfer to 250 ml centrifuge bottles and cool on ice.

4. Add 60 ml of ice-cold 0.2 M NaOH, 1% SDS, mix well by rolling tubes and leave on ice for 5 mins exactly.

5. Add 30 ml ice-cold 3 M KHAc pH 4.8. Mix well by gently rolling tubes and leave for 15 minutes on ice. Finally shake vigorously.

6. Spin at 5,000 rpm (in Sorvall RC-3B) or 10,000 rpm (in Sorvall RC-5B, GSA rotor) for 15 mins at 4oC. Then filter supernatant through two layers of cheese cloth (sterile gauze) into fresh 250 ml bottles.

If the supernatant is not clear then mix solution well and repeat the centrifugation.

7. Precipitate nucleic acid by the addition of 75 ml of isopropanol (~0.6 vols) and centrifuge immediately at 5,000 rpm for 20 min in Sorvall RC-3B or 10,000 rpm, 10 min in Sorvall RC-5B, GSA rotor, setting temperature to 22oC.

8. Pour off supernatant and discard. Dissolve pellet in 10 ml of 100 mM Tris HC1, 10 mM EDTA pH 8.3 (10x TE) and transfer to 50 ml Oakridge tube or 30ml disposable tube.

Add 50 ul of RNAase A (20 mg/ml) and leave 30 min at 37oC.

Add 1 ml 3 M NaCl (or NaAc).

9. Add 10 ml of phenol (0.1% hydroxyquinoline) saturated with 0.3 M TNE, cap tube, shake well and centrifuge for 15 min at 5000 rpm in Sorvall RC-3B (blue adaptors) or 10,000 rpm, 5-10 mins in Sorvall RC-5B, HB4 rotor.

10. Remove upper phase into fresh tube and if necessary repeat (9) once or twice, until no interphase is seen after centrifugation.

11. Precipitate nucleic acid with 20 ml cold EtOH, mix well and leave at -20oC for 30 mins.

12. Spin tube at 10,000 rpm for 15 min at 4oC in Sorvall RC-5B, HB4 rotor. Pour off supernatant and discard, leaving tube to drain for a minute.

At this stage the DNA may be redissolved for separation on a EtBr-CsCl gradient.

If it is advantageous to remove excess RNA, something that a CsCl gradient does only poorly, the following steps may be used.

13. To remove bulk of RNA by differential precipitation, redissolve pellet in 10 ml 0.3 M NaAc ~pH 7 at R/T. Add 5.6 to 5.8 ml R/T isopropanol dropwise on whirlimix. Leave 10 min at R/T then centrifuge for 15 mins at 10,000 rpm, R/T in Sorvall RC-5B, HB4 rotor. (Retain S/N in case plasmid not precipitated.)

14. Wash with 10 ml of R/T 0.3 M NaAc/isopropanol (1:0.6 v/v), spin for 5 min as in (13).

15. Discard supernatant, add 10 ml chilled EtOH and centrifuge again as in (14) but setting temperature to 4oC.

16. Discard supernatant and leave tube to drain for a few minutes. The plasmid may now be dissolved for immediate use or for further purification. About 500 ug to several mg of plasmid (100 ug for cosmids) can be expected from a 500 ml to 1 litre culture.

 

ISOLATION OF MINI-PREP PLASMID DNA FOR SEQUENCING

Primer annealing:

Take 5-8 ul of the plasmid prep., add 8-11 ul water and 4 ul l N NaOH/0,1 mM EDTA, mix and incubate for 5 minutes at 80-99 oC, we usually use 99oC. Add 60 ul ETOH and 2,2 ul 2M ammonium acetate, pH4.6. Vortex and store at -20 oC >30 minutes or O/N, (or l5 minutes in dry ice/ethanol bath). Spin l0 minutes and vacuum-dry the pellet. Resuspend in l0 ul water, add 2 ul annealing buffer and 2 ul primer. Proceed according the instructions of the sequencing kit.

Alternative rapid denaturation & annealing:

5ul DNA, 1ul (EXACTLY) 1N NaOH/0.1mMEDTA and 1 to 5pmol primer, 10ul total volume. Heat at 80-99oC for 5min, (earlier protocols used 22 or 65 oC). Add (EXACTLY) 4 ul TDMN and leave at R/T for 10 minutes. Use directly in sequencing reaction as annealed template.

TDMN:

0.28M TES (free acid)

0.12M HCl

0.05M DTT

0.08M MgCl2

0.2M NaCl

Note: prepare this and the 1M NaOH solution with maximum precision, i.e. using graduated flasks.

Ref: Jones, D.S. and Schofield, J.P. NAR vol. 18, No. 24, 7463-64 (with some modifications)

 

FURTHER PLASMID PURIFICATION

A. On Sucrose Gradients

This method is good for removal of small molecular weight RNA and high molecular weight E. coli DNA. The plasmid recovered is usually pure enough for most enzymes.

1. The plasmid from a 1 litre culture is dissolved in 0.5 ml TE.

2. 16 ml 30% sucrose in TE (autoclaved 20 min, 15 psi) is placed in front chamber of gradient mixer and 18 ml 10% sucrose in TE (also autoclaved) in back chamber.

3. gradient is then pumped into an SW28 centrifuge tube in about 10 min, allowing sucrose to run down wall of tube and overlayer.

4. Tube is transferred to rotor bucket and balanced against a second gradient by removal of some of the upper sucrose layer. The buckets and rotor are brought to the centrifuge and the plasmid over layered onto the gradient just before assembling rotor.

5. Gradients are spun at 23,000 rpm for 15 hours at R/T in Beckman centrifuge, SW28 rotor. (O.K. for 3 to 12 kb plasmids).

6. Gradients are fractionated by displacement with 60% sucrose in TE (should not be autoclaved) in Isco. 0.5 min fractions are taken at pumping speed of 3 ml/min and full scale deflection of 2.0 O.D. (0.5 cm cell is standard but check). Base line must be set up using H2O or TE and lamp allowed to warm up for 10 min before this setting made.

7. Expect:



Either

8a. 3 ml of pooled fractions into polyalomel tube for SW28 rotor. Add 1.1 ml of 3 M NaCl and 6.9 ml TE, mix, then fill tube with cold EtOH and mix again. Leave more than 30 min or O/N at -20oC.

8b. Dialyse pooled fractions against 2 changes ~100 volumes each, of TE, over a period of 3 hrs at 4oC. Finally transfer to polyalomel centrifuge tube (either for SW28 or SW40 rotor) and precipitate with 2 volumes cold EtOH mixing well. Leave more than 30 min or O/N at -20oC.

9. Recover DNA by centrifuging at 25,000 rpm (SW28 rotor) or 35,000 rpm (SW40 rotor) and 4oC for 30 min. Pour off supernatant, fill tube with cold 80% EtOH and spin again for 15 min. Again pour off, supernatant and leave tubes to drain for a few minutes.

10. Vacuum dry for 5 min and redissolve in TE, ~500 ul or at 1 ug/ul.

 

B. On Caesium Chloride-Ethidium Bromide Gradients

This method gives very pure plasmid DNA as far as enzymes are concerned. Separation of super-coiled form from nicked circle form and E. coli DNA is very good, but very little purification from low molecular weight RNA is obtained as this tends to spread throughout gradient.

The following is for 10ml or 5ml of final volume.

1. Plasmid from a 1 litre culture is dissolved in ~7ml of TE, 500ml culture; ~3.5ml.

2. A sterile polypropylene 14 ml tube is placed on a mg accuracy top-pan balance and exactly 8 g (or 4 gm) CsCl weighed into it. This is followed, still on the balance, by the 7 ml (3.5 ml) of plasmid solution and the weight of liquid added made up to 7.5 g (3.75 g) with TE. Wearing disposable gloves 0.5 g (0.25 g) of 10 mg/ml EtBr is added, the tube sealed with parafilm and the CsCl dissolved thoroughly.

3. Dissolved solution (10ml or 5 ml) is transferred to either one or two 5ml Quickfit tube(s) (for VTi65 rotor) or to one 13 ml Quickfit tube (for 70.1Ti rotor) and topped up with paraffin oil using a syringe. Weights of tubes are checked to be within 0.1 g. Seal tubes using the Quickfit sealer.

4. 5ml tubes are centrifuged in VTi65 for 4 hrs at 60krpm or O/N at 45krpm at 22oC. The 13ml tubes are centrifuged at 54krpm for 16 hours, or at 35,000 rpm for more than 48 hours, at 22oC in a 70.1 Ti rotor. Lower speeds give best physical separation as gradient is shallower. Equilibrium at the lower rpm could be achieved faster by spinning for 1 or more hours at the higher rpm then slowing to lower rpm.

5. Tubes are removed from rotor and placed one by one above the 360 nm UV source.

Expected:



6. Piercing top of Quickfit tube with 18 gauge syringe needle allows air to enter the tube during extraction. The super-coiled (sc) zone is extracted from tube by side puncture. A 1 ml syringe (with 18 gauge needle) is used to puncture the tube just below the sc zone and the zone drawn into the syringe. NOT more than 1 ml should be taken.

7. Transfer this 1 ml of supercoiled plasmid to a 4 ml polypropylene tube.

8. Add 1 ml of isoamyl alcohol and shake well. Remove upper phase and discard. Repeat this step once, twice or more times until most of the EtBr is removed from plasmid.

9. Transfer plasmid to polyalomel SW28 centrifuge tube or 30 ml polypropylene tube. Add 10 ml TE, mix, then fill tube with cold EtOH, mix again and leave at -20oC for more than 30 min. but not O/N.

10. Recover plasmid by centrifuging at 25,000 rpm for 30 min and 4oC in Beckman, SW28 rotor or at 10,000 rpm for 30 min and 4oC in Sorvall RC-5B, HB4 rotor. Pour off supernatant, fill tube with cold 80% EtOH and spin again for 15 min. if necessary. Again pour off supernatant and leave tubes to drain for a few minutes.

11. Vacuum dry for 5 min, (NOT for cosmids, which should be just left to drain) and redissolve in TE - about 500 ul for plasmids, 100 ul for cosmids (~1 ug/ul).