Protocols

17.12.01

Work in laminar flow cabinet Before working in laminar flow cabinet; these preparations should be done at least 15 min before start of work in laminar flow cabinet 1) 2) 3) 4)

Switch on laminar flow cabinet Open window of laminar flow cabinet Clean working surface with desinfectant (1% Ultrasol F) Place required materials and solutions into laminar flow cabinet

After working in laminar flow cabinet 1) Clean working surface with desinfectant (1% Ultrasol F) 2) Close the window 3) Switch it all off If working surface is still clean after completion of work, then simply close window of laminar flow cabinet and switch it off.

Protocols

17.12.01

Cloning Preparation of competent cells 1) Obtain 5 ml O/N-culture of E. coli DH5α (or alternative strain) in LB-medium 2) Pour the 5 ml O/N-culture into 400 ml LB-medium Work under laminar flow cabinet! 3) Grow at 37°C to an OD590 of 0.375 All following steps are to be done at 4°C. As work is done on bench top, clean working surface with desinfectant (1% Ultrasol) and switch on Bunsen burner to obtain some sterility. Centrifugation is always done in swinging rotor. Do not use break for deceleration. 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17)

Aliquot culture into 8 x 50 ml Falcon tubes Leave for 10 min on ice Centrifuge for 7 min at 3000 rpm (1600 g) at 4°C (do NOT use brake for deceleration!) Pour off supernatant Carefully resuspend each pellet in 10 ml ice-cold CaCl2-solution Centrifuge for 5 min at 2500 rpm (1100 g) at 4°C (do NOT use brake for deceleration) Discard supernatant Carefully resuspend each pellet in 10 ml ice-cold CaCl2-solution Leave for 30 min on ice Centrifuge for 5 min at 2500 rpm (1100 g) at 4°C (NO brake for deceleration) Discard supernatant Carefully resuspend each pellet in 2 ml ice-cold CaCl2-solution Leave for 1 h on ice Aliquot in 200 µl volumes (sufficient for 6 transformations per aliquot; use prechilled tubes) 18) Store competent cells at -80°C

Protocols

17.12.01

Cloning Assessment of the transformation efficiency of competent cells 1) Mix in 1.5 ml Eppendorf tube: 10 ng of pGEM-vector (2 µl of a 5 ng/µl concentrated stock solution) 100 µl competent cells 2) Incubate for 20 min at 4°C 3) Incubate for 2 min at 42°C 4) Incubate for 5 min at 4°C 5) add 1 ml prechilled SOC-medium 6) Incubate for 45 min at 37°C (rotator incubator: 250 rpm) 7) Prepare 1:10 and 1:100 dilution using additional SOC-medium 8) plate it out 100 µl of the three different concentrations (1:1; 1:10; 1:100) on agar-plates containing Carbenicillin 9) let colonies grow O/N at 37°C 10) check number of colonies Aim: transformation efficiency of 106 (106 colonies per µg of transformed DNA) 0.01 µg pGEM were used; thus, we expect a total of 104 transformed colonies (in 1 ml SOC) Therefore, the following colony counts refer to the following transformation efficiencies: 100 colonies in the 1:1 dilution (100 µl of the original solution) 107 100 colonies in the 1:10 dilution (10 µl of the original solution) 106 100 colonies in the 1:100 dilution (1 µl of the original solution) 105

Protocols

17.12.01

Cloning Pouring of Agar-Plates 0) Before starting: a) thaw an aliquot of Carbenicillin (about 820µl) (25 mg/ml) on ice b) prepare laminar flow cabinet as described c) required materials for laminar flow cabinet: • petri dishes, labelled on side of the bottom plate (Initials + LAC + date) • 1 ml pipette tips • 1 ml pipette 1) 2) 3) 4) 5) 6) 7)

Microwave 200 ml of LA-medium (heat= 250; 12 min) let medium cool down to RT in laminar flow cabinet add 820 µl of Carbenicillin Pour Agar-plates (200 ml of medium should result in about 10 plates) let the medium settle down (about 15-30 min) place lids on petri dishes turn the petridishes upside down in order to avoid condensating water to drop from the lids onto the Agar 8) store the plates at 4°C 9) Switch off laminar flow cabinet; clean if necessary (see instructions for laminar flow cabinet)

Protocols

17.12.01

Cloning Ligation Using Promega’s pGEM-T Vector System All steps are to be done at 4°C 1) Thaw on ice: i) pGem-vector ii) aliquotted T4 Ligase buffer iii) purified PCR product 1) Set up ligation reaction mix: 25-100 ng DNA 5 µl (2.5µl) 2X T4 Ligase buffer 1µl (0.5µl) pGEM-vector 1µl (0.5µl) T4 Ligase ....µl millipore H2O (total volume 10µl (5µl)) 1) Incubate at 4°C (10°C) for 15h 2) Thereafter, ligation-mix can be used directly for transformation of bacteria

Transformation 1) thaw an aliquot of competent E. coli DH5α on ice 2) pipet into 1.5 ml Eppendorf-tube: 30 µl competent E. coli DH5α 2.5 µl of the ligation-mix 1) 2) 3) 4) 5) 6)

Incubate for 20 min at 4°C Incubate for 2 min at 42°C Incubate for 5 min at 4°C add 150 µl prechilled SOC-medium Incubate for 45 min – 1 h at 37°C (rotator incubator: 250 rpm) plate it out on an agar-plate containing Carbenicillin, IPTG, X-Gal

Protocols

17.12.01

Cloning Selection of recombinants on Agar-plates Steps 1-3 can be done in laminar flow cabinet; required materials: X-GAL and IPTG on ice 200 µl pipette tips 200 µl and 20 µl pipette agar plates sterile drigalski spatula (sterilised through heat outside laminar flow cabinet!) The remaining steps are done on bench top. 0) Before starting a) Clean working area with desinfectant (1% Ultrasol) b) Switch on Bunsen burner to obtain some sterility c) Get Agar plates out of the fridge and leave them at RT to warm up d) Label agar plates on bottom with sample name 1) Apply to an Agar-plate: 40 µl X-GAL (20 mg/ml) 8 µl IPTG (100 mg/ml) 1) Distribute it evenly 2) Leave it to dry for about 15-30min 3) Sterilise drigalski spatula using heat; sterilisation must be repeated after each sample 4) Add 80-100 µl of transformed bacteria 5) Distribute it evenly using sterile dirgalski spatula 6) Leave it to dry for about 10-30 min 7) Incubate at 37 °C overnight (about 15-18 hours) 8) Check for positive recombinants (white colour)

Protocols

17.12.01

Cloning Amplification’ of recombinants in LB-medium Steps 1-4 to be done in laminar flow cabinet! Required materials: 15 ml Falcon tubes (labelled) + rack sterile tooth picks LB medium Carbenicillin, thawed on ice 1 ml pipette tips 1 ml pipette 1) Add to 200 ml LB-medium: 820 µl Carbenicillin (25 mg/ml) 1) for each positive colony, prepare a sterile 15 ml Falcon tube with: 5 ml of LB-medium including Carbenicillin 1) Pick-up part of a positive colony using a sterile tooth-pick (either from a colony grown on an Agar-plate or from a glycerol ‘stock’) 2) transfer cells into LB-Medium 3) Incubate at 37°C overnight (for about 18 h) in a rotator (200 rpm)

Long-term storage of recombinants: Preparation of Glycerol-‘stocks’ Work in laminar flow cabinet! Required materials: 86% Gylcerol 1.5 ml Eppendorf tubes (labelled) + rack 1 ml pipette tips 1 ml pipette O/N culture in rack 1) Add to a 1.5 ml Eppendorf-tube: 200 µl 86% Glycerol 300 µl of an overnight culture from a positive colony 1) briefly invert the tube 2) store at -80°C

Protocols

17.12.01

Cloning PCR-assay of cloning success Steps 3 and 4 are done in laminar flow cabinet; required materials: PCR tubes in rack Agar plates with white colonies Sterile tooth picks 1) for each ‘white’ colony, set-up a PCR (25 µl reaction volumes) 2) Primers to be used: pUC/M13-Forward (pUCF) and pUC/M13-Reverse (pUCR) 3) after PCR-master mix has been prepared and distributed to the different tubes, pick up part of a positive (white) colony using a sterile tooth-pick 4) transfer these cells to the PCR-reaction mix 5) use the following PCR-profile (Name: CLONE): Initial denturation: 2:00 min 25 cycles: 0:20 min 1:00 min 1-2:00 min Final extension: 5:00 min

95°C 95°C 52°C 72°C 72°C

1) Check the results via Agarose-gel-electrophoresis