DH10bac大腸桿菌表達(dá)宿主菌，感受態(tài)細(xì)胞

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DH10Bac

菌株抗性：(bom+, tra-, mob-)

菌株特點(diǎn)：F- mcrA Δ(mrr-hsdRMS-mcrBC)φ80dlacZΔM15 ΔlacX74deoR recA1endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsL nupG /bMON14272 /pMON7124

Preparation ofDH10BAC competent cells

Stock solutions

LBMedium: Autoclave 1O g tryptone, 5 g yeast extract, and 5 g NaCl in 1 L.

CaCl2solution: 60mM CaCl2 15%(v/v) glycerol, 10mM PIPES, pH adjusted to 7.0 withKOH. Filter sterilize.

Protocol

1.Inoculatea single colony of E.coli DH10BAC into 5mls of LB medium containing tetracyclineat 10 μg/ml and kanamycin at 50 μg/ml. Grow overnight at 37°C with shaking (250rpm).

2.Inoculate4 mls of the culture into 400ml LB medium containing tetracycline at 10 μg/mland kanamycin at 50 μg/ml in a 2 litre flask. Grow at 37°C with shaking (250rpm)to an OD590 of 0.375. (This procedure requires that cells be growing rapidlyie. early or mid log phase).

3.Aliquotculture into 8 x 50ml pre-chilled, sterile polypropylene tubes and leave thetubes on ice 5 to 10mins. Centrifuge cells at 4000xg, 4°C for 15 mins.

4.Resuspend(gently) each pellet in 10ml ice cold CaCl2 solution. Centrifuge 15 mins at4000xg, 4°C.

5.Resuspendeach pellet in 10mls cold CaCl2 solution. Keep resuspended cells on ice for30mins. Centrifuge 15 mins at 4000xg, 4°C.

6.Resuspendeach pellet completely in 2mls of ice cold CaCl2 solution. Aliquot 250 μl intoprechilled, sterile polypropylene tubes. Freeze immediately at –70°C.