Protocol

Abbreviation

SmallScaleExpression_protocol_Toronto

Name

SmallScaleExpression_protocol_Toronto

Laboratory name

University of Calgary

Type

expression

Description

Small scale expression

DAY 1
1.Place PCR plate containing the plasmids (3 µl at 50-100ng/ul) on ice ~10 min.
2.Add 25-30 µl of competent BL21-CodonPlus(DE3)-RIL cells (Stratagene).
3.Leave on ice for 10 min.
4.Heat shock @ 42°C for 45 sec.
5.Place on ice for 2 min.
6.Recover in 850 µl of SOC for 1 h @ 37°C with shaking.
7.Spin down @ 3000 rpm for 10 min.
8.Discard supernatant (flip plate only, do not tap, ~100 µl of media should remain at bottom). Use 55 µl to plate on LB + ampicillin (plasmid antibiotic resistance 100 µg/ml) + chloramphenicol (20 µg/ml) (keep remaining cell suspension at 4oC in case the cells don’t grow). Use glass beads to spread the sample.
9.Incubate plates overnight @ 37°C. Remove beads.

NOTE: Plates with transformations should be used within two weeks, maximum.


DAY 2
1.Take one colony from a FRESH transformation plate and aseptically transfer into 5 ml of autodinduction-Studier media (ZYP-5052) containing antibiotics in a 30 ml culture tube.
2.Incubate at 37oC for 4-5 hours with shaking (220 rpm) until OD600 ~2 is reached.
3.Switch temperature to 20oC for overnight incubation (12-14 h).
4.Gels can be cast on this day.



DAY 3
1.Transfer 500 µl of culture into 1.5 ml microtubes, spin at 14 000 rpm for 1 min, and discard the supernatant.
2.Freeze pellet in liquid nitrogen and thaw by incubating at room temperature. If testing 96 samples, keep half at -80oC until ready to test (or if you have more than you can handle, keep those you are not testing at -80oC).
3.Suspend pellet in 500 µl of Lysis Buffer and rotate (put 4-5 microtubes in a 50 ml Falcon tube and put on rotator in cold room; Power 65%) for 1.5 h at 4oC.
4.Take 40 µl of lysate and mix to 10 µl of 5X Sample Buffer for SDS-PAGE (Total Expression) in a PCR plate.
Two possibilities for the layout of the samples in the plates. You can use one plate per fraction (one for total and one for soluble) or use one plate per set of samples tested (top lanes for total and bottom lanes for soluble). There is no problem to reheat samples from a previous day.
5.Spin lysate at 14 000 rpm for 15 min.
6.Take 40 µl of supernatant and add 10 µl of 5X Sample Buffer for SDS-PAGE (Soluble Fraction) in a PCR plate.
7.Incubate plate 10 min at 95oC in PCR cycler.
8.Load 10 µl of sample per well with multichannel (1 well will separate each sample), which gives the possibility to have Total Expression and Soluble Fraction side by side for each clone.
9.Load 8 µl of ladder.
10.Run SDS-PAGE at 140 V for 70 min.
11.Open cassette with «scraper».
12.Wash gel in water for 3x for 5min at room temperature with shaking.
13.Discard water and add Invitrogen SimplyBlue SafeStain (#LC6065)for staining the gel(s), this is be be done at room temperature with shaking.
14.Remove stain and rinse for 1hr with water(you may want to repeat this step).
15.Scan the gel(s) and dry (Gel-Dry Drying Solution from Invitrogen #LC4025).

RECIPES


ZYP-5052 rich medium for auto-induction
*Everything is sterile*
dH2O 828 ml
1000X Me mix 1 ml
1 M MgSO4 1 ml
10X TY 100 ml
GGL 20 ml
20X NPS 50 ml

Add Me Mix to water first. If not, it will precipitate. Add 1 ml of ampicillin stock and 0.5 ml of chloramphenicol stock. (Do not autoclave)


1000X trace Metal mixture (Me Mix)
0.1 M FeCl3•6H2O 50 ml
1 M CaCl2 2 ml
1 M MnCl2•4H2O 1 ml
1 M ZnSO4•7H2O 1 ml
0.2 M CoCl2•6H2O 1 ml
0.1 M CuCl2•2H2O 2 ml
0.2 M NiCl2•6H2O 1 ml
0.1 M Na2MoO4•5H2O 2 ml
0.1 M Na2SeO3•5H2O 2 ml
0.1 M H3BO3 2 ml

Add ~36 ml of dH2O (to complete to 100 ml) and filter sterilize. Store at 4oC until use. When making growth media, add the metal mix in the water first to avoid precipitation.


Stock solutions for Me Mix
Only the FeCl3 solution is made in 50 mM HCl. All others are done in dH2O. Everything is kept at RTo.





10X TY
Tryptone 100 g
Yeast extract 50 g

Complete to 1 L with dH2O and autoclave. Keep at RTo.



GGL
Glycerol 250 g
Glucose 25 g
α-lactose 100 g

Lactose takes a while to dissolve. Complete to 1 L with dH2O and autoclave. Keep at RTo.


20X NPS
(NH4)2SO4 66 g
KH2PO4 136 g
Na2HPO4 142 g

Complete to 1 L with dH2O and autoclave. Keep at RTo.


Stock Ampicillin (100 mg/ml)
1 g of ampicillin in 10 ml dH2O.
Filter sterilize (0.2 µm), aliquot and store at -20°C. Avoid freeze/thaw cycles.


Stock Chloramphenicol (50 mg/ml)
0.5 g of chloramphenicol in 10 ml of ethanol. Aliquot and store at -20oC.



1X cell lysis buffer non-denaturing
1 M Tris-HCl pH 7.5 20 ml
5 M NaCl 30 ml
0.2 M Na2EDTA 5 ml
0.8 M EGTA 1.25 ml
Triton X-100 10 ml
1 M Sodium pyrophosphate 2.5 ml
1 M β-glycerophosphate 1 ml
1 M Na3VO4 1 ml
Leupeptin 1 mg
dH2O

Complete to 1 L with dH2O. Keep at 4oC until use.


Stock solution for cell-lysis buffer non-denaturing
All solutions are made in water (special protocol for Na3VO4) and kept at 4oC. Only leupeptin is kept at -20oC in its powder form.


1 M Na3VO4
sodium orthovanadate 4.6 g
dH2O 15-20 ml

Adjust to pH 10, no less (can be a little above). Boil until translucent and readjust the pH. Repeat these steps until solution remains clear at pH 10. Complete to 25 ml with dH2O. Aliquot and store at -20oC or keep at 4oC. Avoid freeze/thaw cycles.