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| - | {{AUC_TURKEY}}
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| - | {{:LGmenu:AUC_TURKEY}}
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| - | <div class=WordSection1>
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| - | <p class=MsoNormal><span style='font-size:12.0pt;line-height:115%'> </span></p>
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| - | <p class=MsoNormal style='margin-bottom:7.2pt;border:none;padding:0cm'><b><span
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| - | style='font-size:16.0pt'>Procedures for LB Agar Preparation</span></b></p>
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| - |
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| - | </div>
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| - |
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| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
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| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
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| - | style='font:7.0pt "Times New Roman"'> </span></span><span
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| - | style='font-size:12.0pt'>In a steril environment, the tare of the container
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| - | should be measured and subtracted from the overall weight.</span></p>
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| - |
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| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
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| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
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| - | style='font:7.0pt "Times New Roman"'> </span></span><span
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| - | style='font-size:12.0pt'>7 grams of LB Agar is put in the container.</span></p>
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| - |
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| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
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| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
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| - | style='font:7.0pt "Times New Roman"'> </span></span><span
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| - | style='font-size:12.0pt'>200 ml distilled water or is put into a graduated
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| - | cylinder.</span></p>
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| - |
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| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
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| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
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| - | style='font:7.0pt "Times New Roman"'> </span></span><span
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| - | style='font-size:12.0pt'>These two are mixed in a beaker.</span></p>
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| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
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| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
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| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>The opening of the beaker is covered with aliminium
| |
| - | folio in such a way that it does not conctact with air.</span></p>
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| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
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| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
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| - | style='font:7.0pt "Times New Roman"'> </span></span><span
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| - | style='font-size:12.0pt'>Autoclave tape is sticked on to the aliminium.</span></p>
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| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
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| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
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| - | style='font-size:12.0pt'>The beaker is placed in to the autoclave machine.</span></p>
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| - |
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| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
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| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Distilled water or demineralized water is put into the
| |
| - | autoclave machine. The water level in the autoclave machine has to be a little
| |
| - | higher than the liquid level of the beaker.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>After closing the lid of the machine, the 90 minute autoclave
| |
| - | process is given start.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Take out the beaker and add antibiotics if required.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal'><i><span style='font-size:12.0pt'>Warnings for the Autoclave!</span></i></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Use only demineralised or disttiled water with the
| |
| - | device.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Do not open the cover until the manometer drops to
| |
| - | zero during the operation.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please do not use the autoclave for other purposes
| |
| - | than sterilization and agar.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please do not use the autoclave to sterilize
| |
| - | explosive, inflammable and oxidizing materials.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please be cautious when you are closing the lid not to
| |
| - | trap your hand.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please beware of the steam exhaust when you are
| |
| - | opening to autoclave after sterilization.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please wear protective gloves before removibg
| |
| - | materials from the chamber. Do not access the chamber unless the vapor exhaust
| |
| - | is finalized.</span></p>
| |
| - |
| |
| - | <div style='border:none;border-bottom:solid #AAAAAA 1.0pt;padding:0cm 0cm 2.0pt 0cm'>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:7.2pt;border:none;padding:0cm'><b><span
| |
| - | style='font-size:16.0pt'>Procedures for LB Broth Preparation</span></b></p>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>In a steril environment, the tare of the container
| |
| - | should be measured and subtracted from the overall weight.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>7 grams of LB Broth is put in the container.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>200 ml distilled water or is put into a graduated
| |
| - | cylinder.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>These two are mixed in a beaker.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>The opening of the beaker is covered with aliminium
| |
| - | folio in such a way that it does not conctact with air.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Autoclave tape is sticked on to the aliminium.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>The beaker is placed in to the autoclave machine.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Distilled water or demineralized water is put into the
| |
| - | autoclave machine. The water level in the autoclave machine has to be a little
| |
| - | higher than the liquid level of the beaker.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>After closing the lid of the machine, the 90 minute
| |
| - | autoclave process is given start.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Take out the beaker and add antibiotics if required.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal'><i><span style='font-size:12.0pt'>Warnings for the Autoclave!</span></i></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Use only demineralised or disttiled water with the
| |
| - | device.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Do not open the cover until the manometer drops to
| |
| - | zero during the operation.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please do not use the autoclave for other purposes
| |
| - | than sterilization and agar.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please do not use the autoclave to sterilize
| |
| - | explosive, inflammable and oxidizing materials.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please be cautious when you are closing the lid not to
| |
| - | trap your hand.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please beware of the steam exhaust when you are
| |
| - | opening to autoclave after sterilization.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Please wear protective gloves before removibg
| |
| - | materials from the chamber. Do not access the chamber unless the vapor exhaust
| |
| - | is finalized.</span></p>
| |
| - |
| |
| - | <div style='border:none;border-bottom:solid #AAAAAA 1.0pt;padding:0cm 0cm 2.0pt 0cm'>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:7.2pt;border:none;padding:0cm'><b><span
| |
| - | style='font-size:16.0pt'>Procedures for Transformation</span></b></p>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Transfer 500 ul LB Broth to 1.5 ml microcentrifuge
| |
| - | tubes. This should be done close to a source of fire to prevent contamination.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Place the microcentrifuge tubes containing LB Broth in
| |
| - | a 42 C heat block for incubation.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Take 1 ul plasmid and place them in 1.5 ml centrifuge
| |
| - | tubes.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Add 50 ul competent cells to the plasmid.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Centrifuge them at 3000 rpm for 20-30 seconds.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Incubate the cells in ice for 45 minutes.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>After 45 minutes, heat the tubes in the 42 C heat
| |
| - | block for a maximum of 90 seconds.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>The same tubes should be placed in ice and should be
| |
| - | incubated for 5 minutes.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Afterwards, 450 ul LB should be added to the cells to
| |
| - | complete them to 500 ul.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>The microcentrifuge tubes are then sticked to the
| |
| - | shaker horizantally and shaked for 1 hour with 320 rpm at 37 C.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>150 ul of the mixture(200 ul for digestion) is then
| |
| - | placed on the plate to spread.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>It is then spread on the plate and the plates are
| |
| - | incubated at 37 C for 16 hours.</span></p>
| |
| - |
| |
| - | <div style='border:none;border-bottom:solid #AAAAAA 1.0pt;padding:0cm 0cm 2.0pt 0cm'>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:7.2pt;border:none;padding:0cm'><b><span
| |
| - | style='font-size:16.0pt'>Procedures for Isolation</span></b></p>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>The LB Media should be transferred to 1.5 ml
| |
| - | centrifuge tubes.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>These tubes are then centrifuged at 13,000 rpm for 10
| |
| - | minutes at room temperature.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>After the centrifuge, the supernatent should be
| |
| - | disposed without taking any pellets along with it.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>The pelleted cells should be suspended in 250 ul
| |
| - | Resuspension Solution and the tubes should be vortexed so that no cell clumps
| |
| - | remain.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>250 ul Lysis Solution is added. The tubes are inverted
| |
| - | 4-6 times but the inversion shouldn't be done really fast as the Lysis Solution
| |
| - | must not be shaken. Also it is smart to heat the Lysis Solution in order to
| |
| - | ensure that no pericipitate remain.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>350 ul Neutralization Solution should be added and the
| |
| - | tube should be inverted immediately and throughly by inverting 4-6 times.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Centrifuge for 5 minutes to pellet cell debris and
| |
| - | chromosomal DNA.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Transfer the supernatent to a spin column without
| |
| - | taking any of the pellets.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Centrifuge the spin column for 1 minutes and discard
| |
| - | the liquid at the bottom. Place the column at the same tube again.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Add 500 ul Wash Solution and centrifuge for 30-60
| |
| - | seconds. Discard the flow-through and place column back in.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Repeat the same process again with 500 ul Wash
| |
| - | Solution.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Centrifuge for an additional 1 minute.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Transfer the columns into 1.5 ml microcentrifuge tubes
| |
| - | and leave their caps open for 2 minutes so that the ethanol that they contain
| |
| - | dissolves in air.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Add 50 ul Elution Buffer to the center of the silica
| |
| - | membrane to elute the plasmid DNA. The pipette tip shouldn't the membrane.
| |
| - | Incubate for 2 minutes and centrifuge for 2 minutes afterwards.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Discard the spin column and store at -20 C.</span></p>
| |
| - |
| |
| - | <div style='border:none;border-bottom:solid #AAAAAA 1.0pt;padding:0cm 0cm 2.0pt 0cm'>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:7.2pt;border:none;padding:0cm'><b><span
| |
| - | style='font-size:16.0pt'>Digestion Protocol</span></b></p>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Take the average of the nucleic acid concentrations
| |
| - | measured by the spectrometer.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Divide 500 by the DNA average.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Add 5ul Ne Buffer.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Add 0.5ul BSA Buffer.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Add 1 ul of the enzymes with barrier tips.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>If you cut with EcoR1 and SpI, it will be up stream.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>If you cut with Xbal and Pst1, it will be down stream.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Subtract the amount of DNA from 42.5 ul. This result
| |
| - | will be the amount of NFW used.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Add the NFW with barrier tips and do one pippetting
| |
| - | while taking the NFW.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Then the DNA is put into the PCR and is left there for
| |
| - | 30 minutes.</span></p>
| |
| - |
| |
| - | <div style='border:none;border-bottom:solid #AAAAAA 1.0pt;padding:0cm 0cm 2.0pt 0cm'>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:7.2pt;border:none;padding:0cm'><b><span
| |
| - | style='font-size:16.0pt'>Ligation Protocol</span></b></p>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>2ul up stream is put into a eppendorf.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>2ul down stream is also added.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>2ul plasmid is mixed in as well.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>2ul Taq Buffer is inserted to the mixture.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>1ul T4 DNA ligase is then added with barrier tips.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>11ul NFW is added with barrier tips and should be
| |
| - | pipetted once.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Then the DNA is put into the PCR and is left there for
| |
| - | 30 minutes.</span></p>
| |
| - |
| |
| - | <div style='border:none;border-bottom:solid #AAAAAA 1.0pt;padding:0cm 0cm 2.0pt 0cm'>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:7.2pt;border:none;padding:0cm'><b><span
| |
| - | style='font-size:16.0pt'>Gel Preparation</span></b></p>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Mix 100ml TAE and 0,8 gram agarose in a glass beaker.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>The mixture is then heated in a microwave for 3
| |
| - | minutes.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Afterwards, 3,6 ul EtBr is added and the beaker is
| |
| - | mixed on a magnetic mixer.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:1.2pt;margin-left:18.0pt;text-indent:
| |
| - | -18.0pt;line-height:14.3pt'><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Mold them and wait for 20 minutes fort he gel to
| |
| - | harden.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-left:18.0pt;line-height:normal; '><b><u><span style='font-size:16.0pt'>Procedures for Western Blot</span></u></b></p>
| |
| - |
| |
| - | <p class=MsoListParagraphCxSpFirst style='text-indent:-18.0pt;line-height:normal;
| |
| - | '><span style='font-size:10.0pt;font-family:Wingdings'>§<span
| |
| - | style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>Western blots allow investigators to determine the
| |
| - | molecular weight of a protein and to measure relative amounts of the protein
| |
| - | present in different samples.</span></p>
| |
| - |
| |
| - | <p class=MsoListParagraphCxSpMiddle style='margin-right:36.0pt;text-indent:
| |
| - | -18.0pt;line-height:normal; '><span style='font-size:10.0pt;
| |
| - | font-family:Wingdings'>§<span style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>1) Proteins are separated by gel electrophoresis,
| |
| - | usually </span><a
| |
| - | href="http://www.bio.davidson.edu/genomics/method/SDSPAGE/SDSPAGE.html"><span
| |
| - | style='font-size:12.0pt;color:windowtext'>SDS-PAGE</span></a><span
| |
| - | style='font-size:12.0pt'>.</span></p>
| |
| - |
| |
| - | <p class=MsoListParagraphCxSpMiddle style='margin-right:36.0pt;text-indent:
| |
| - | -18.0pt;line-height:normal; '><span style='font-size:10.0pt;
| |
| - | font-family:Wingdings'>§<span style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>2) The proteins are transfered to a sheet of special
| |
| - | blotting paper called nitrocellulose, though other types of paper, or
| |
| - | membranes, can be used. The proteins retain the same pattern of separation they
| |
| - | had on the gel.</span></p>
| |
| - |
| |
| - | <p class=MsoListParagraphCxSpMiddle style='margin-right:36.0pt;text-indent:
| |
| - | -18.0pt;line-height:normal; '><span style='font-size:10.0pt;
| |
| - | font-family:Wingdings'>§<span style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>3) The blot is incubated with a generic protein (such
| |
| - | as milk proteins) to bind to any remaining sticky places on the nitrocellulose.
| |
| - | An antibody is then added to the solution which is able to bind to its specific
| |
| - | protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish
| |
| - | peroxidase) or dye attached to it which cannot be seen at this time.</span></p>
| |
| - |
| |
| - | <p class=MsoListParagraphCxSpLast style='margin-right:36.0pt;text-indent:-18.0pt;
| |
| - | line-height:normal; '><span style='font-size:10.0pt;font-family:
| |
| - | Wingdings'>§<span style='font:7.0pt "Times New Roman"'> </span></span><span
| |
| - | style='font-size:12.0pt'>4) The location of the antibody is revealed by
| |
| - | incubating it with a colorless substrate that the attached enzyme converts to a
| |
| - | colored product that can be seen and photographed.</span></p>
| |
| - |
| |
| - | <p class=MsoNormal><span style='font-size:16.0pt;line-height:115%'> </span></p>
| |
| - |
| |
| - | <p class=MsoNormal><b><u><span style='font-size:16.0pt;line-height:115%'>Procedures
| |
| - | of Sonication</span></u></b></p>
| |
| - |
| |
| - | <p class=MsoNormal><span style='font-size:12.0pt;line-height:115%'> </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>1) Resuspend pellet of
| |
| - | 10ml cell culture in 1ml lysis buffer (or 100ml bacterial culture for very low
| |
| - | expression level).</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'> </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>Suggested Lysis buffer
| |
| - | : 140mM NaCl; 2.7mM KCL; 10mM Na2HPO4; 1.8mM KH2PO4; pH 7.3 (PBS) </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>
| |
| - |
| |
| - | or 100mM NaCl; 25mM TrisHCl; pH 8.0 </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>
| |
| - |
| |
| - | optional 0.02% NaN3 (azide) </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>
| |
| - |
| |
| - | optional protease inhibitors</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'> </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>Optional additives to
| |
| - | the lysis buffer </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>a) 1mM PMSF or protease
| |
| - | inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from
| |
| - | Sigma) </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>b) Dnase 100U/ml or
| |
| - | 25-50ug/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of
| |
| - | 10mMMgCl2. </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>c) Lysozime 0.2mg/ml.
| |
| - | Incubate 10min 4°C. </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>d) ßME, DTT or DTE
| |
| - | up to 10mM for proteins with many cysteines. </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>e) 0.1-2% Triton X-100,
| |
| - | NP40; or any other detergent that do not affect the biological activity of your
| |
| - | protein. </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>f) 10% glycerol (for
| |
| - | stabilization of the protein and prevention of aggregation).</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'> </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>2) Sonicate in ice
| |
| - | bucket 3 x 10sec or more if the cells are not completely disrupted (Lysis is
| |
| - | complete when the cloudy cell suspension becomes translucent. Avoid protein
| |
| - | denaturation by frothing).</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'> </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>3) Spin 5min 13000rpm
| |
| - | 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies
| |
| - | proteins (pellet). Use supernatant for next step. Keep sample of 40ul of supernatant
| |
| - | for PAGE-SDS and Western blot: soluble proteins</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'> </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
| |
| - | normal; '><span style='font-size:12.0pt'>4) Resuspend pellet in
| |
| - | another 1ml lysis buffer and keep sample of 40ul for PAGE-SDS and Western blot:
| |
| - | insoluble proteins, or unlysed cells. </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><b><u><span
| |
| - | style='font-size:16.0pt'>Procedures for Competent Cell Preparation</span></u></b></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><b><u><span
| |
| - | style='font-size:16.0pt'> A. Preparing glassware and media eliminate detergent </span></u></b></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>1. Autoclaving glassware filled 3/4 with DD-H2O to
| |
| - | remove most detergent residue </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>2. Media and buffers in detergent free glassware and
| |
| - | cultures grown up in detergent free glassware </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><b><u><span
| |
| - | style='font-size:16.0pt'>B. Preparation of the competent cells </span></u></b></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>Reagents: </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-Glycerol stock </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-LB plate </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-MgCl2-CaCl2 solution </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-MgCl2-CaCl2 solutuion </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-MgCl2</span><span style='font-size:12.0pt;font-family:
| |
| - | "MS Gothic"'>‧</span><span style='font-size:12.0pt'>6H2O 3.25g </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-CaCl2</span><span style='font-size:12.0pt;font-family:
| |
| - | "MS Gothic"'>‧</span><span style='font-size:12.0pt'>2H2O 0.6g </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>Add H2O to 200ml </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-100mM CaCl2 </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-100mM CaCl2 solution </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-CaCl2</span><span style='font-size:12.0pt;font-family:
| |
| - | "MS Gothic"'>‧</span><span style='font-size:12.0pt'>2H2O 2.95g </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>Add H2O to 200m; </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>-80% glycerol </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:16.0pt'>-Liquid nitrogen </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><b><span
| |
| - | style='font-size:16.0pt'>DAY1:</span></b></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>1. Flame the metal inoculating loop until it is red
| |
| - | got and then cools it down </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>2. Scrape off a portion from the top of the frozen
| |
| - | glycerol stock [DO NOT THAW]</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>3. Streak it onto the LB plate</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>4. Put the stock back to -80C immediately</span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>5. Leave the plates for 5 minutes and place them
| |
| - | upside down in the 37C incubator for 16-20 hours </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><b><span
| |
| - | style='font-size:16.0pt'> DAY 2</span></b></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>6. Pick a single colony into 5ml of LB medium </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>7. Inoculate the culture overnight at 37C with shaking
| |
| - | at 250rpm </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:16.0pt'> <b>DAY3</b></span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>8. Inoculate 100ml LB medium with 1ml of saturated
| |
| - | overnight culture </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>9. Shake at 37C until OD600=0.4 (usually 2-3 hours) </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>10. Place in an ice bath for 10 minutes [ After this
| |
| - | point the cells should never </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>touch anything that is warm] </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>11. Pre-cool solution, centrifuge, pipette tips,
| |
| - | falcon, eppendorf </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>12. Transfer the culture into two pre-chilled 50ml
| |
| - | falcon </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>13. Centrifuge at 2700x g for 10 minutes at 4C </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>14. Remove the medium, resuspend the cell pellet with
| |
| - | 1.6ml ice0cold </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>100mM CaCl2 by swirling on ice gently </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>15. Incubate on ice gor 30 minutes </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>16. Centrifuge at 2700x g for 10 minutes at 4C </span></p>
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| - |
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| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>17. Remove the medium, resuspend the cell pellet with
| |
| - | 1.6ml ice-cold 100mM CaCl3 by swiriling on ice gently </span></p>
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| - |
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| - | <p class=MsoNormal style='line-height:normal; '><span
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| - | style='font-size:12.0pt'>18. Incubate on ice for 20 minutes </span></p>
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| - |
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| - | <p class=MsoNormal style='line-height:normal; '><span
| |
| - | style='font-size:12.0pt'>19. Combine cells to one tube and add 0.5 ml ice-cold
| |
| - | 80% glycerol and swirl to mix </span></p>
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| - |
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| - | <p class=MsoNormal style='line-height:normal; '><span
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| - | style='font-size:12.0pt'>20. Freeze 100ul aliquots in liquid nitrogen </span></p>
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| - |
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| - | <p class=MsoNormal style='line-height:normal; '><span
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| - | style='font-size:12.0pt'>21. Store in -80C </span></p>
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