组织的分离实验_EDTA 消化分离法 分析行业新闻
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- Desalt DNA template by EtOH precipitation in NaOAc followed by at least 2x washes with 70% EtOH. Resuspend in 5 - 15 µL of sterile H2O.
- Rinse cuvettes (if they have been used before) 5x with deionied H2O, and place them on ice. This is sufficient to avoid background growth in most cases.
- Set a BioRad MicroPulser to "Ec1" for 1 mm cuvettes, or "Ec 2" for 2 mm cuvettes.
- Electroporate the DNA into the bacteria:
- Add 5 µL of DNA to 50 µL of bacteria and mix by pipetting.
- Transfer bacteria/DNA mix to a cold cuvette, and immediately pulse in the electroporator.
- Quickly add 1 mL of r.t. L.B. to the bacteria in the cuvette. Use a sterile Pasteur pipette to transfer the suspension to a bacteria tube.
- Repeat for additional DNA samples and control DNA.
Rotate at 37ºC x30 min. to allow the bacteria to recover. Meanwhile plate LB/Amp plates with IPTG + X-gal if blue/white selection of colonies will be performed, as follows: - Mix 100 µL X-gal + 20 µL of IPTG per plate.
- Spread X-gal/IPTG mix across surface of plate with a sterile glass spreader and a plate spinner.
- Allow the mix to infiltrate the media for 20 min.
Perform two 10-fold serial dilutions of the LB/bacteria suspension into fresh sterile L.B. Concentrate the remaining bacteria by spinning 1 min. at 5k rpm in a sterile microcentrifuge tube, and then resuspend pellet in 0.1 mL of L.B. Plate 100 µL of the various concentrations of bacteria onto LB/Amp (+ X-gal/IPTG as necessary), and spread with a sterile spreader and plate spinner to evenly coat the plate. Record the DNA construct and the Bacteria Dilution Factor (9x, 1x, 1/10x, 1/100x) on each plate. Incubate the plates inverted at 37ºC o.n. They may need to be placed in a container if the humidity of the incubator is so low that it causes the agar to dry out. Remove the plates when the colonies are ~1mm in diameter. The color development on X-gal treated plates will continue to occur after the plates are removed from the incubator. Pick white colonies (also with no central blue coloration) and restreak on L.B./Amp plates to ensure that pure clones are obtained before performing plasmid preps. Store at 4ºC for at least 1 hr. (to firm up the agar) if colony hybridiation will be performed. Seal edges of plates with parafilm for storage up to 1 week. Count the numbers of colonies on the plate with control DNA to determine the efficiency of competent cells: - Efficiency (Transformants/µg) = colonies/plate x (Bacterial dilution factor) x 20,000
- Highly competent cells should have ~50 colonies on the plate with a 1/10 dilution of the control DNA's bacteria suspension.
Materials
- Control DNA (e.g. pBS) diluted to 1 pg/µL in sterile H2O, on ice.Electrocompetent bacteria, frozen (50 - 100 µL aliquots), on ice.BioRad cuvettes (1 mm gap), on ice. Sterile L-Broth (L.B.) Bacteria culture tubes
- P1000, P200, P20 Pipetman and sterile tipsSterile Pasteur pipets and bulb.37ºC incubator and rotating wheelL.B./Amp bacterial culture plates (or other suitable selection media)
- X-gal 20 mg/mL in DMF (dimethlyformamide), store at -20ºCIPTG 0.2 g/mL in H2O, store at -20ºC
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