SLAC protein sequence: MAPAAKGITARTAPAGGEVRHLKMYAEKLADGOMGYGFEKGKASVPGPLIEVN EGD
ID: 187200 • Letter: S
Question
SLAC protein sequence: MAPAAKGITARTAPAGGEVRHLKMYAEKLADGOMGYGFEKGKASVPGPLIEVN EGDTLHIEFTNTMDVRASLHVHGLDYEISSDGTAMNKSDVEPGGTRTYTWRTHK PGRRDDGTWRPGSAGYWHYHDHVVGTEHGTGGIRNGLYGPVIVRRKGDVLPAT HTIVFNDMTINNRKPHTGPDFEATVGDRVEIVMITHGEYYHTFHMHGHRWADRT GILTGPDDPSRVIDNKITGPADSFGFQIIAGEGVGAGAWMYHCHVQSHSDMGMV GLFLVKKPDGTIPGYEPHEHGGATAKSGESGEPTGGAAAHEHEH With His-tag wt: 36 kD, Extinction coefficient: 43890 cm M Can you please write the procedures for the incubation and autoclave? 1. Large scale SLAC purification First day: . Incubate overnight SLAC BL21 from frozen stock into 2 x 5 mL LB/Amp at 37 oC. .Autoclave 100 mL LB in 500 mL Erlenmeyer flasks.Explanation / Answer
Incubation: Protein epressing BL21 E.coli cells ( SLAC BL21) is taken out from the frozen stock ( generally frozen in minus 80 degree). The E.coli stock solution ( normally 100-200 µl) is added to a LB medieum which contains AMP antibiotic ( LB/ Amp). This medium is taken in 5ml conical flask and SLAC BL21 will be added to it. Next this flask will be kept in a incubator for overnight with a temperature setting of 37 degree. This will help to grow SLAC BL21 E,coli cells.
Autoclave: autoclave is used to sterilize the freshly prepared media or equipments neessary for a particular experiments. Autoclave is done on autoclave machine which is basically a pressure chamber and sterilization is done by steam at high pressure. Stem at high pressure kills the bacteria, spores and germs which can not be killed or eradicated by clsning, boiling water or detergent.
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