Difference between revisions of "Team:SKLMT-China/Material"

REAGENTS

QIAquick Gel Extraction Kit (Qiagen, cat. no. 28704)

QIAquick PCR Purification Kit (Qiagen, cat. no. 28104)

Qiagen Plasmid Mini Kit (Qiagen, cat. no. 12123)

Tryptone (Oxoid, cat. no. LP0042)

Yeast extract (Oxoid, cat. no. LP0021)

NaCl (Sangon Biotec, cat. no. SB0476)

Agar (Solarbio, cat. no. A8190)

NaOH (Sigma-Aldrich, cat. no. S8045)

Autoclaved ddH2O kept at room temperature (RT, 18–22 °C) and on ice

Sodium acetate trihydrate (NaOAc•3H2O; Sinopharm, cat. no. 10018718)

Lysozyme (Sigma-Aldrich, cat. no. 62970)

Proteinase K (Roche, cat. no. 03115879001)

Trizma base (Sigma-Aldrich, cat. no. T6066)

Na2EDTA (Sigma-Aldrich, cat. no. E5134)

HCl (Sinopharm, cat. no. 10011008) Sodium dodecyl sulfate (SDS; Sigma-Aldrich, cat. no. L3771)

Phenol–chloroform–isoamyl alcohol mixture (25:24:1; Sigma-Aldrich,cat. no. 77617)

Isopropanol (Sinopharm, cat. no. 80109218)

Ethanol, absolute (Sinopharm, cat. no. 10009218)

70% (vol/vol) ethanol

RNase A (10 mg ml − 1; DNase and protease free; Thermo Scientific,cat. no. EN0531)

l-Arabinose (Sigma-Aldrich, cat. no. A3256)

l-Rhamnose (Sigma-Aldrich, cat. no. 83650)

1-kb DNA ladder (New England BioLabs, cat. no. N3232)

Restriction enzymes (New England BioLabs)

PrimeSTAR Max DNA Polymerase (Takara, cat. no. R045B)

10× Tris/boric acid/EDTA (TBE) buffer (Bio-Rad, cat. no. 161-070)

Ethidium bromide solution (10 mg/ml; Dingguo, cat. no. NEP028-1)

Agarose (Takara, cat. no. 5261)

Buffer P1 (Qiagen, cat. no. 19051)

Buffer P2 (Qiagen, cat. no. 19052)

Buffer P3 (Qiagen, cat. no. 19053)

EQUIPMENT

Benchtop centrifuge, kept at 2 °C (Eppendorf, model 5424R,cat. no. 5404000.014)

Benchtop centrifuge, kept at RT (Eppendorf, model 5424R,cat. no. 5424000.010)

Centrifuge (Eppendorf, model 5810R, cat. no. 5811000.398)

Electroporation cuvettes with 1-mm gap, kept on ice (Eppendorf,cat. no. 940001005)

Electroporator (Eppendorf, model 2510, cat. no. 940000009)

Thermomixer (Eppendorf, model F1.5, cat. no. 5384000.071)

MixMate (Eppendorf, cat. no. 022674226)

Vortex (Scientific Industries, cat. no. G560E)

50-ml centrifuge tubes (sterile, DNase/RNase-Free; Sangon Biotec,cat. no. CT788-GS)

Petri dishes, 94 mm × 16 mm (Greiner Bio-One, cat. no. 633180)

Digital gel imaging system (GelDoc XR + , Bio-Rad)

UV spectrophotometer (NanoDrop 2000c, Thermo Scientific)

UV-visible spectrophotometer (T6 New Century, Purkinje General Instrument)

Syringe needle, 25-gauge 5/8, 0.5 mm × 16 mm (BD Medical, cat. no. 301805)

Multipette (Eppendorf, cat. no. 4981000.019)

10-ml Combitips (Eppendorf, cat. no. 0030089.464)

Gel electrophoresis apparatus (Beijing Junyi, cat. no. JY300C)

Incubators kept at 30 and 37 °C (Ningbo Jiangnan, cat. no. HWS-0288)

Sterile 10-ul inoculation loop (Sangon Biotec, cat. no. IL311-10-S-Q)

Sterile 1-ul inoculation loop (Sangon Biotec, cat. no. IL311-1-S)

0.2-um syringe filter (Pall, cat. no. PN4612)

Self-made tissue rollers or cotton swabs (available from most grocery stores)

37 °C and 50 °C water baths

REAGENT SETUP

LB medium: For 1 liter, dissolve 10 g of tryptone, 5g of yeast extract and 5g of NaCl in ~800 ml of ddH2O, adjust the pH to 7.0 (adjust the pH to 8.0 with NaOH for low-salt LB) and bring the final volume to 1 liter. Transfer 100-ml aliquots to a glass stock bottle and autoclave. The medium can be kept at RT for several months. Add antibiotics before use.

LB agar plates: Transfer 200-ml aliquots of LB medium to a glass stock bottle, add 2.4 g of agar and autoclave the medium. Pour molten medium into sterile Petri dishes. If antibiotics are required, add them when the autoclaved medium has cooled to 50 °C. Plates should dry for at least 15 min in a sterile hood with lid off to reduce condensation. Before use, remove prepared plates from the refrigerator and keep them at RT for at least 10 min to avoid condensation and cold shock to the cells.

0.8% (wt/vol) Agarose gels: For 100 ml, add 0.8 g of agarose to 100 ml of 1× Tris/boric acid/EDTA (TBE) buffer. Completely melt the agarose gel in a microwave and then cool it to ~60 °C. Add 5ul of ethidium bromide solution (10 mg ml−1), mix it thoroughly, pour the gel into a mold with an appropriate comb and let it solidify. The agarose gels should be freshly prepared before use.

3 M Sodium acetate pH 7.5: For 100 ml, dissolve 40.8 g of sodium acetate trihydrate in ~80 ml of ddH2O, and bring it to a final volume of 100 ml. The pH should be ~7.5 without the addition of acetic acid. Transfer the solution to a glass stock bottle and autoclave. The solution can be stored at RT for at least 12 months.

10% (wt/vol) l-Arabinose and 10% (wt/vol) l-rhamnose: For 50 ml, dissolve 5 g of sugar powder in ~40 ml of ddH2O and bring it to a final volume of 50 ml. Pass the solution through 0.2-um syringe filters to sterilize it inside a sterile laminar hood. Prepare 1-ml aliquots in sterile 1.5-ml tubes. The stock solution can be stored at − 20 °C for at least 12 months.

1× TBE electrophoresis buffer: Dilute 10× TBE buffer in ddH2O to a 1× working solution, and store it at RT for several months.

10% (wt/vol) SDS: For 100 ml, add 10 g of SDS to ~80 ml of autoclaved ddH2O. Stir until SDS is dissolved, and bring it to a final volume of 100 ml. Transfer the solution to a glass stock bottle. The solution can be stored at RT for at least 12 months.

Proteinase K (20 mg ml − 1): We usually purchase 100-mg vials of ProteinaseK. Add 5 ml of autoclaved ddH2O to each 100-mg vial to make a final concentration of 20 mg ml − 1. The solution can be stored in the vials at − 20 °C for several months.

1.0 M Tris-HCl (pH 8.0): For 1 liter, add 121.1 g of Trizma base to ~800 ml of ddH2O. Stir until dissolved. Adjust the pH to 8.0 with concentrated HCl and bring it to a final volume of 1,000 ml. Transfer the solution to glass stock bottles and autoclave. The solution can be stored at RT for at least 12 months.

10 mM Tris-HCl (pH 8.0): For 1 liter, add 10 ml of 1.0 M Tris-HCl (pH 8.0) to 990 ml of autoclaved ddH2O. Stir to mix the contents and transfer the solution to a glass stock bottle. The buffer can be stored at RT for at least 12 months.

0.5 M EDTA (pH 8.0): For 1 liter, add 186.1 g of Na2EDTA to ~800 ml of ddH2O. Stir until the contents have dissolved. Adjust the pH to 8.0 with NaOH (~20 g pellets) and bring to a final volume of 1,000 ml. Transfer the solution to glass stock bottles and autoclave. The solution can be stored at RT for at least 12 months.

5 M NaCl: For 100 ml, dissolve 29.2 g of NaCl in ~80 ml of ddH2O, and bring to a final volume of 100 ml. Transfer the solution to a glass stock bottle and autoclave it. The solution can be stored at RT for at least 12 months.

SET (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 8.0): For 100 ml, add 1.5 ml of 5 M NaCl, 5 ml of 0.5 M EDTA (pH 8.0) and 2 ml of 1.0 M Tris-HCl (pH 8.0) to 91.5 ml of autoclaved ddH2O. Stir to mix and transfer the solution to a glass stock bottle. The buffer can be stored at RT for at least 12 months.

antibiotics

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