Handling and storage of clones

Handling and storage condition vary depending of additive antibiotics. The colour of a lid is corresponded to antibiotics. Additive antibiotic is corresponded to Clone ID.

Clone ID* with BH or BN prefix [ex. 6316691 (BH20-H12)、5379725 (BN3-D6)］

Clones are cultured on LB agar medium (50 ?g/ml Ampicillin）. Store at 4°C without freezing. Pick single colonies of the clone off of a tube and grow 24 hours in the same composition of medium as soon as possible.

Clone ID* with BG prefix [ex. 6877960 (BG1-F10)］

Clones are cultured on LB agar medium （27 ?g/ml Chloramphenicol). Store at 4°C without freezing. Pick single colonies of the clone off of a tube and grow 24 hours at 30°C in the same composition of medium as soon as possible.

※Clone ID is an original ID of Source BioScience and different from IMAGE ID nor MGC ID. Clone ID is indicated on the tube.

Culture more than ten single colonies of the each clone in the LB liquid medium with the same antibiotic and 8% glycerol, and freeze the glycerol stock at -70°C. The clone stocks are ready to culture again during the plasmid purification process.

The single colony picking, as same as a library replication, is an essential steps for storage and handling of clones to avoid risks of contamination, cross contamination, unexpected mutation of original clones. With larger number of single colonies, larger possibility to recover the original clone. In the same way, you can isolate the original clone from a contaminated clone stock.

Handling and storage

Handling and storage condition vary depending of additive antibiotics. Additive antibiotic is corresponded to Clone ID.

IRBH (Xenopus laevis), and IRBN (Xenopus tropicalis)

Clones are replicated in liquid LB agar medium（50 ?g/ml Ampicillin) with 8% glycerol. Plated are shipped with dry ice. Store them at -70°C immediately. To replicate clones from the plate, culture at 37°C an overnight with the same medium.

IRBG (Xenopus laevis)

Clones are replicated in the liquid LB medium（27?g/ml Chloramphenicol） with 8% glycerol. Plated are shipped with dry ice. Store them at -70°C immediately. To replicate clones from the plate, culture at 30°C an overnight with the same medium.

Freeze stockedclones are ready to culture to prepare plasmid and PCR amplification.

Clones on the same plate are on the same vector.

→IRBH (Xenopus laevis)

→IRBG (Xenopus laevis)

→IRBN (Xenopus tropicalis)

See I.M.A.G.E. vector information for more information.

→I.M.A.G.E.Vector information

□Plasmid DNA mini-prep

Normal mini-prep is suitable. Source BioScience recommends the following protocol.

• Spread the clone on to LB agar plate to culture an overnight at 37°C (amp) or 30°C (CM or KM).
• Dispens 5 ml of LB medium to a sterilized 50 ml micro tube and add an appropriate antibiotic. Inoculate a single colony to the medium to shaking culture (170rpm) an overnight at 37°C (amp) or 30°C (CM or KM).
• [For glycerol stock, dispense 140 ul of the medium to each 1.5 ml miro tubes and add 40 ul of 80% glycerol.]
• Put 2 ml of medium into 2 ml micro tube and is centrifuged 15 min, 3000rpm.
• Throw away the supernatant, and add 200 ul of GTE solution to suspend the pellet. Flowingly, add 5 ul of RNase A stock (10mg/ml) and allow to stand at room temperature
• After Add 400 ul of 0.2M NaOH/1% SDS solution that was prepared immediately before use and mix by inverting, allow stand on ice for 5 min.
• Add 300?l of 3M KAc and mix by inverting, and allow stand on ice for 10 min.
• Centrifuge 10 min, 13000rpm, and fractionat the supernatant into 1.5 ml micro tube with 1 ml of chilled ethanol. Mix with a Vortex mixer and allow stand on ice for 30 min.
• After centrifuged 10 min, 13000rpm, throw the ethanol away carefully (suction method is desirable).
• Add 200 ul of chilled ethanol, and mix with a Vrtex mixer, and them centrifuge 10 min, 13000rpm to throw away ethanol carefully as step 9.
• After let the pellet dry, add 50 ul of TE to keep it at 4°C an overnight.

□Solution

10ml GTE:

0.5ml 20% glucose

0.25ml 1M Tris/HCl pH7.5

0.2ml 0.5M EDTA pH8.0

9.05ml Sterile water

100ml 0.2M NaOH/1% SDS:

0.8 g NaOH

5ml 20% SDS

95ml Sterile water

□Plasmid DNA purification with PEG (recommended to perform before sequence analysis)

• Add the sama amount of 20％PEG/2.5M NaCl solution to the plasmid DNA solution and allow stand at ambient temperature 5-10 min, then Centrifuge 10-15 min, 13000rpm.
• Throw away the supernatant and wash the obtained pellet with 70% ethanol. After letting the pellet dry, dissolve it into 20 ul of sterile water.
• To confirm yield of DNA, add loading buffer to 5 ul of the solution and perform electrophoresis with an appropriate DNA marker.

□PCR purfication

Inserted DNA amplification by Using PRC with an appropriate primer. Purification of PCR product with spin column or sAP/exo1 （shrimp alkaline phosphatase/exonuclease1）priore to sequencing is essential.

□References

• Werle. E, Schneider. C, Renner. M, Volker.M and Fiehn. W. (1994) Convenient single-step, one tube purification of PCR products for direct sequencing. Nucl. Acids Res. 22: 4354-4355.
• Hanke. M and Wink. M. (1994) Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. BioTechniques 17: 858-860.