Bacterial transformation & infection *
STANDARD E. COLI TRANSFORMATION *
Stock Solutions: *
Competent Cells:- *
RAPID TRANSFORMATION OF E. COLI *
TRANSFORMATION OF E. COLI USING RBCL *
Stock Solutions *
Frozen storage of competent cells *
PREPARATION OF IN VITRO l PACKAGING MIXES and PACKAGING. *
Stock Solutions *
STANDARD E. COLI TRANSFORMATION
50 mM CaCl2
|100 mM Tris HC1 pH 7.5||1 ml 1 M|
|100 mM CaCl2||1 ml 1 M|
|100 mM MgCl2||1 ml 1 M|
|Final volume||10 ml|
1. Streak HB101 onto L-broth plate and grow at 37oC overnight.
2. Single colony looped into 1 ml. Incubate at 37oC with shaking overnight.
3. Dilute 1:100 in Erlenmeyer flask and incubate at 37oC with vigorous shaking for 3-4 hours until OD 600 nm (1 cm) = 0.6 (2 x108 cells/ml).
4. Centrifuge at 3,000 rpm for 10 min, and 4oC in Sorvall RC-3B.
5. Resuspend cell pellet in 1/2 volume ice cold 50 mM CaCl2. Stand for 15 mins on ice (1 hour if cells to be frozen).
6. Centrifuge 3,000 rpm for 10 min, and 4oC in Sorvall RC-3B.
7. Resuspend in 1/10th volume ice cold 50 mM CaCl2, or resuspend in 20% glycerol, 50 mM CaCl2, pool, aliquot and freeze at -70oC.
The following steps should be done in polypropylene tubes, more usually sterile eppendorfs or 4 ml Falcon snap cap tubes.
8. Incubate 100 ul bacteria, 10 ul 10x TCM, 90 ul DNA (maximum of 100 ng> DNA must not be in EDTA or be over 50 mM NaCl), for 30 mins on ice, 2 mins at 37oC and 10 mins at room temperature.
9. Add 1 ml L-broth prewarmed to 37oC.
10. Incubate 2 mins at 42oC, 1 hour at 37oC.
11. Spin down 10 min, 3000 rpm and resuspend cell pellet in 100 ul L-broth. Spread directly onto appropriate antibiotic L-broth plate.
12. Incubate overnight at 37oC.
Efficiency: 1-3 x106 colonies/ug of sc pAT153 DNA.
RAPID TRANSFORMATION OF E. COLI
After Pope & Kent, (1996, NAR 24 p536-537) with modification.
1. Mix 1 to 12 ul of DNA solution (1 to 10 (or more?) ng) with 100ul of competent cells (from standard protocol).
2. Leave on ice for 1 to 180 min, apparently not important.
3. Plate onto appropriate ampicillin or tetracyclin LB plates which have been prewarmed to 37oC and taken from incubator immediately before use (this is the key to success!).
4. Incubate O/N at 37oC.
The method was originally performed on cells made competent by incubation in 100mM CaCl2 for 30 min on ice and stored at 4oC or, with addition of 10% glycerol, at -70oC. Efficiency in excess of standard procedure was claimed but depended on the concentration of NaCl in the DNA solution. This should be kept below 20mM and preferably should be 1mM or less.
Protocol works very well with our standard competent cells, see "STANDARD E. COLI TRANSFORMATION".
TRANSFORMATION OF E. COLI USING RBCL
(C.M. Read, after D. Hanahan 1983, J. Mol. Biol. 166, 557-580)
2% x/v Bacto® Tryptone (Difco)
0.5% w/v Bacto® Yeast extract (Difco)
10 mM NaCl
2.5 mM KC1
10 mM MgCl2
10 mM MgSO4
Medium prepared without Mg2+ and autoclaved. Prior to use add 10 ml of 2 M Mg2+ solution (autoclaved) per litre of broth.
2 M Mg2+ solution:
1 M MgCl.6H2O
1 M MgSO4.7H2O
SOB medium plus 20 mM glucose.
10 mM K-MES pH 6.20 (Sigma)
100 mM RbCl (Alpha 88688)
45 mM MnCl2.4H2O
10 mM CaCl2.2H2O
3 mM Hexamine Cobalt (III) Chloride (Alpha 23144)
1 M MES is adjusted to pH 6.3 with KOH and autoclaved. TFB buffer is autoclaved.
Transformations carried out with TFB buffer containing 100 mM KC1 rather than RbC1 appear to be just as efficient.
DMSO (alpha 13778):
Sealed bottle of DMSO is opened and contents portioned out as 500 ul in 500 ul Eppendorfs and stored at -20oC. Single portion is thawed and used on a given day and discarded.
2.25 M DTT
40 mM K-Acetate pH 6.0
Sterile filtered and placed in 500 ul Eppendorfs at -70oC.
|1% w/v Bacto Tryptone (Difco)||10 g|
|0.5 w/v Bacto Yeast extract (Difco)||5 g|
|1.5% Bacto Agar (Difco)||15 g|
|10 mM NaCl||0.50 g|
|10 mM MgSO4.7H2O||see below|
|Final volume||1 l|
Medium prepared without Mg2+ and autoclaved. Prior to use add 10 ml of 1 M MgSO4 solution (autoclaved) per litre of medium.
Ampicillin used at 35 ug/ml. Tetracyclin used at 12.5 ug/ml, with Mg2+ omitted from medium.
FSB buffer (for frozen storage of competent cells):
10 mM K Acetate
100 mM KC1
45 mM MnCl2.4H2O
10 mM CaCl2.2H2O
3 mM Hexamine CoCl3
10% glass distilled glycerol (BRL 5514)
1 M K Acetate is adjusted to pH 7.0, sterile filtered and stored at -20oC. pH of complete solution is adjusted to pH 6.4 with HCl, autoclaved and stored at 4oC.
1. Streak DH1 or HB101 onto LM plates. Incubate O/N at 37oC.
2. Colonies picked off plate and dispersed in 1 ml of SOB medium by gentle vortexing (require one 2.5 mm diameter colony/10-15 ml broth).
3. Use this to inoculate a prerinsed flask containing 50 ml of SOB medium (in 500 ml flask) (require 2.5 ml of culture/transformation). Incubate at 37oC, 225 rpm until OD 550 nm (1 cm) = 0.5 (about 2-2.5 hours.).
4. Collect cells in 50 ml tubes (eg Falcon 2070), place on ice for 10-15 mins, then centrifuge at 3000 rpm, 10 mins, 4oC in Sorvall RC-3B.
5. Resuspend cells in 1/3 vol. TFB, place on ice for 10-15 mins, then centrifuge at 3000 rpm, 10 mins, 4oC in Sorvall RC-3B.
6. Resuspend cells in 1/12.5 of the original volume (i.e. 2.5 ml culture to 200 ul) in TFB.
7. Add 7 ul fresh DMSO/200 ul culture in TFB, swirl and leave on ice for 5 mins.
8. Add 7 ul DTT solution/200 ul culture, swirl and leave on ice for 5 mins.
9. Add another 7 ul DMSO/200 ul culture, swirl and leave on ice for 5 mins.
10. 200 ul aliquots placed into chilled 17x100 mm tubes (Falcon 2006 or 2059). Add DNA in less than 10 ul (DNA in 1/2x TE), swirl and leave on ice for 30 mins.
11. Place mixture at 42oC for one and a half min, then on ice for 1-2 mins.
12. Add 800 ul SOC medium (~20oC) and incubate at 37oC, 225 rpm for 1 hour.
13. Appropriate fraction of culture pipetted onto LM plate containing antibiotic and incubate at 37oC. If more than 10% is to be plated then add 2 ml of SOC after the incubation, pellet cells at 3000 rpm, 10 min, 20oC in Sorvall RC-3B and resuspend in 100 ul of SOC. Then spread directly on plate.
If less than 10% is to be plated, then make up to 100 ul with SOB medium before plating.
Efficiency: 1-3 x108 colonies/ug of sc pAT153 DNA.
Frozen storage of competent cells
1. Cells prepared as described in steps 1-6, but using FSB buffer.
2. Add 7 ul fresh DMSO/200 ul culture in FSB, swirl and leave on ice for 5 mins.
3. Add another 7 ul DMSO/200 ul culture, swirl and leave on ice for 5 mins.
4. Portion out cells in 200 ul aliquots into Falcon 2006 or 2059 tubes. Freeze quickly in liquid N2 and store at -70oC.
1. Remove tubes and thaw in air at 20oC.
2. When just liquid place on ice for 10 mins. Add DNA and continue protocol (steps 10-).
PREPARATION OF IN VITRO l PACKAGING MIXES and PACKAGING.
(C.M. Read, modified Blattner method)
Note:- Commercial packaging mixes are nowadays much more efficient, but for some routine work the following may be of use.
|20 mM Tris HCl pH 8.0||400 ul of 1 M stock|
|3 mM MgCl2||60 ul of 1 M stock|
|0.05% v/v mercaptoethanol||10 ul|
|1 mM EDTA pH 7.5||40 ul of 0.5 M stock|
|Final volume||20 ml|
|6 mM Tris HCl pH 7.5||30 ul, 1 M|
|60 mM Spermidine chloride (neutralised with Tris base)||3 ml, 0.1 M|
|18 mM MgCl2||90 ul, 1 M stock|
|15 mM ATP (Boehringer, neutralised with NH4OH)||300 ul, 250 mM|
|30 mM mercaptoethanol||1 ul, 14M|
|Final volume||5 ml|
10 mM Tris pH 7.4
100 mM NaCl
10 mM MgCl2
Cold 10% Sucrose, 50 mM Tris HCl pH 7.5
Fresh 2 mg/ml Lysozyme (Sigma) in 250 mM Tris HCl pH 7.5.
1. From slopes or HMFM cultures streak BHB 2688 and BHB 2690 onto L-broth plates (2 of each) and incubate one at the permissive temp, 30-32oC, the other at the non-permissive temp, 42oC.
If a significant number of colonies grow at 42oC as compared with 32oC, then:
(i) take single colonies from the 32oC plate and streak in duplicate onto separate sectors of two L-broth plates. Incubate one plate at 30-32oC, other plate at 42oC.
(ii) Select a sector from the 32oC plate which does not show any growth on the 42oC plate and take a loop of bacteria into 10 ml L-broth and shaking O/N at 30-32oC (do the BHB 2690 first).
1. Inoculate 500 ml of L-broth with 5 ml of an O/N culture of BHB 2690. Grow at 30-32oC for ~4 hours.
2. At 2-3 x108 cells/ml (OD 600 nm (1 cm) = 0.3) induce by placing bottle for 15 mins without shaking in water bath at 45oC. Then add ice to bring temp down to 37oC, leave for 10 mins.
3. Grow at 37-38oC, shaking hard for 1 hour.
4. Check if bacteria are induced:
A drop of chloroform added to a small sample of culture should make the culture go clear in 1 minute (use glass tube).
5. Cool on ice.
6. Harvest in 2x 250 ml bottles, spinning at 9,000 rpm for 10 min, 4oC in Sorvall RC-5B, GSA rotor.
7. Remove all supernatant and resuspend each pellet in 0.5 ml buffer A. Pool and transfer to single plastic tube (15 ml Falcon tube).
8. Dilute with 2.6 ml buffer A.
9. Sonicate without foaming. 10-20 3s blasts necessary at max power with at least 1 minute between blasts. (Keep below 10oC). About 50 ul of suspension should be spun in eppendorf centrifuge for 2 mins and continue sonication until almost no pellet is visible. Solution should be no longer viscous.
10. Transfer to 4 ml polypropylene tube. Pellet debris at 6,000 rpm, 6 min, 4oC in Sorvall RC-5B, HB4 rotor (can spin harder. Pellet should be small).
Remove supernatant and aliquot 50 ul into precooled (on ice) Eppendorf tubes.
11. Quick freeze in liquid N2. Store at -70oC.
Freeze Thaw Lysate
1. Inoculate 3x 500 ml of L-broth in 2 litre flasks with 3x 5 ml of an O/N culture of BHB 2688.
2. Grow at 30-32oC for about 4 hours.
3. At 2-3 x108 cells/ml (OD 600 nm (1 cm) = 0.3) induce lambda by placing bottles for 15 mins without shaking in water bath at 45oC. Then add ice to bring temperature down to 37oC and leave for 10 min.
4. Grow at 37oC shaking hard for 1 hour.
Check if bacteria are induced as before.
5. Harvest in 6x 250 ml bottles by spinning at 9,000 rpm, 10 min. at 4oC on Sorvall RC-5B, GSA rotor.
6. Drain off all supernatant by pouring off the bulk and letting tube rest at an angle on ice with pellet upwards. After a couple of minutes remove all drained liquid.
7. Resuspend each pellet in 0.5 ml of cold 10% sucrose, 50 mM Tris pH 7.5. Pool all supernatants and dispense into 2x 10 ml tubes (suitable for MSE 10x 10 ml Ti rotor).
8. Add 75 ul fresh lysozyme solution to each tube. Mix gently, but well.
9. Quick freeze in liquid N2. Store at -70oC O/N.
10. Pre-cool MSE 10x 10 ml Ti rotor.
11. Thaw at room temperature and then at 4oC until pellet completely thawed. Cool on ice.
12. Add 75 ul buffer Q1 to each tube. Mix gently but well.
13. Spin at 35,000 rpm for 35 min at 4oC on MSE Superspeed 65.
14. Remove supernatant, and aliquot 50 ul into pre-cooled Eppendorf tubes. Quick freeze in liquid N2 and store at -70oC.
In Vitro Packaging Reaction and Transduction
1. Day 1:- Inoculate 100 ul of ED8767 from HMFM into 10 ml L-groth+0.8% maltose. Incubate at 37oC O/N.
2. Day 2:- Thaw FTL and SE on ice. Mix the following in this order:
7 ul Buffer A
1-2 ul DNA (up to 2 ug)
1 ul Buffer Q1
3.5 ul SE
5 ul FTL
Whirlimix and incubate for 1 hour at 25oC.
3. O/N culture of ED8767 spun down at 3,000 rpm, 15 min and 20oC in Sorvall RC-3B. Drain off supernatant.
Resuspend cells in 5 ml of 10 mM MgCl2.
4. Add 250 ul aliquots to each packaging reaction, vortex and leave at R/T for 15 mins to transduce.
Add 2 ml of L-broth (prewarmed to 37oC) and then incubate without shaking for 30 mins at 37oC to express drug resistance.
5. Remove appropriate fraction, make up to 100 ul with L-broth and plate on antibiotic L-broth.
If plating all: Spin down cells at 3000 rpm, 10 min, 20oC in Sorvall RC-3B. Resuspend cells in 100 ul L-broth and plate.
Incubate inverted plates at 37oC O/N.