Team:KIT-Kyoto/Notebook/YJL
From 2013.igem.org
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<h2>YJL068C</h2> | <h2>YJL068C</h2> | ||
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+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>13th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We designed the | ||
+ | PCR primers to amplify<i> YJL068C</i> gene.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>19<sup>th</sup> | ||
+ | August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt'><span | ||
+ | lang=EN-US style='font-size:11.0pt'>1.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:11.0pt'>Make Primer mix</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We added 102.2 µL | ||
+ | and 100.3 µL H<sub>2</sub>O to the forward and reverse primers, respectively, to | ||
+ | make 100 pmol/µL solutions.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We obtained 10 µL | ||
+ | of each primer solution and added 80 µL H<sub>2</sub>O</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> to make the | ||
+ | 10 pmol/µL primer mix.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt'><span | ||
+ | lang=EN-US style='font-size:11.0pt'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:11.0pt'>PCR</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We performed PCR | ||
+ | to amplify the <i>YJL068C</i> gene.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=162 valign=top style='width:121.45pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=162 valign=top style='width:121.45pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>50µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=162 valign=top style='width:121.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>dNTP</span></p> | ||
+ | </td> | ||
+ | <td width=162 valign=top style='width:121.45pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>20µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=162 valign=top style='width:121.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Primer mix</span></p> | ||
+ | </td> | ||
+ | <td width=162 valign=top style='width:121.45pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=162 valign=top style='width:121.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Genome DNA</span></p> | ||
+ | </td> | ||
+ | <td width=162 valign=top style='width:121.45pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>0.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=162 valign=top style='width:121.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>KOD-FX</span></p> | ||
+ | </td> | ||
+ | <td width=162 valign=top style='width:121.45pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=162 valign=top style='width:121.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>H<sub>2</sub>O</span></p> | ||
+ | </td> | ||
+ | <td width=162 valign=top style='width:121.45pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>26.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> (Annealing : | ||
+ | 52°C)</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We detected two | ||
+ | DNA bands around 1400bp and 3000bp.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>(The correct band | ||
+ | is 1400bp.)</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We changed | ||
+ | annealing temperature to 54°C and 57°C and tried PCR again.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We detected a | ||
+ | correct size DNA band around 1400bp.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>The clear DNA band | ||
+ | was amplified at 54˚C (annealing temperature).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We performed PCR | ||
+ | to amplify <i>YJL068C</i> gene at 54°C of annealing temp (total volume was 500 µL).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>20<sup>th</sup> | ||
+ | August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We performed PCR | ||
+ | to check whether the primers can work or not.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>(Annealing temperature: | ||
+ | 55°C)</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We detected a DNA | ||
+ | band of the correct size around 900bp.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We purified the <i>YJL068C</i> | ||
+ | gene (PCR was performed on 8/19).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested <i>YJL068C</i> | ||
+ | with <i>Ban</i>II to check whether PCR succeeded or not.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We ligated <i>YJL068C</i> | ||
+ | and pSB1C3.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>24µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>M Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xba</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Spe</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:41.65pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>6µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>H Buff.</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:41.65pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>H<sub>2</sub>O</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:41.65pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>11µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Ban</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>II</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:41.65pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested pSB1C3 | ||
+ | with <i>Xba</i>I and <i>Spe</i>I at 37°C for 90 minutes.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pSB1C3</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>49µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>M Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>5.78µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xba</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Spe</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We added 1µL BAP | ||
+ | to dephosphorylate the <i>pcb1c3</i> gene.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We electrophoresed | ||
+ | the <i>YJL068C</i> gene<i> </i>and pSB1C3 (digested for ligation) in 1% Blue | ||
+ | gel.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We saw the clear | ||
+ | band of YJL068C, and not so clear band of pSB1C3.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>They were | ||
+ | extracted from the Blue gel.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We prepared the spare | ||
+ | the spare in case of failure (made stocks of purified YJL068C and pSB1C3).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We purified | ||
+ | YJL068C and pSB1C3 (these were in one tube) and ligated them.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We transformed it | ||
+ | all (20 µL) into <i>E.coli</i> cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <span lang=EN-US style='font-size:11.0pt;font-family:"Century","serif"'><br | ||
+ | clear=all style='page-break-before:always'> | ||
+ | </span> | ||
+ | |||
+ | <p class=MsoNormal align=left style='text-align:left'><span lang=EN-US | ||
+ | style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>21th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We checked the <i>YJL068C</i> | ||
+ | gene (digested with <i>Ban</i>2 8/20) by electrophoresing.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We could see | ||
+ | correct band (succeeded PCR).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We checked of the | ||
+ | insert by colony cracking (transformed 8/20).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We couldn’t see | ||
+ | clear bands, but cultured furthermore.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We made the spare | ||
+ | in case of failure (prepared 8/20).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We purified the <i>YJL068C</i> | ||
+ | gene (digested 8/20).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested pSB1C3 | ||
+ | with <i>Xba</i>I and <i>Spe</i>I (90 minutes at 37°C), and added 1µL BAP to it.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pSB1C3</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>96µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>11µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xba</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Spe</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We electrophoresed | ||
+ | pSB1C3 it in 1% Blue gel.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>It was extracted | ||
+ | from the Blue gel.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=114 valign=top style='width:85.45pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=114 valign=top style='width:85.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pSB1C3</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=114 valign=top style='width:85.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Ligation Mix</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>10µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We added 5 µL H<sub>2</sub>O | ||
+ | to dried <i>YJL068C</i> gene and pSB1C3 each.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We ligated them | ||
+ | and transformed it into <i>E.coli </i>cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>22th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We did miniprep to | ||
+ | purify DNA.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested <i>YJL068C</i> | ||
+ | in pSB1C3 with <i>Pst</i>I and <i>Eco</i>RI, and incubated at 37°C for 90 | ||
+ | minutes.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Plasmid DNA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>10µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>H Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>H<sub>2</sub>O</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>6µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Eco</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>RI</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Pst</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We applied them to | ||
+ | 1% agarose gel electrophoresis and checked them.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>5 samples showed the | ||
+ | correct size.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested pSB1C3 | ||
+ | with <i>Bsi</i>WI.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=107 valign=top style='width:80.1pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pSB1C3</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>10µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=107 valign=top style='width:80.1pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>NEB Buffer 3</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=107 valign=top style='width:80.1pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Bsi</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>WI</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=107 valign=top style='width:80.1pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>H<sub>2</sub>O</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>7µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>23th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We applied <i>YJL068C</i> | ||
+ | in pSB1C3 (prepared on 8/22) to 1% agarose gel electrophoresis.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We detected the | ||
+ | correct size of DNA.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Each transformant | ||
+ | was cultured in LB liquid medium with chloramphenicol.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Plasmid DNA was | ||
+ | purified by miniprep.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>24th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We added 29.5µL of | ||
+ | H<sub>2</sub>O to purified YJL068C.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested <i>YJL068C</i> | ||
+ | with <i>Ban</i>II and checked.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:41.65pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>H Buff.</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:41.65pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>H<sub>2</sub>O</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:41.65pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>12µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Ban</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>II</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:41.65pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We detected the | ||
+ | correct size of DNA band.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested<i> | ||
+ | YJL068C </i>with <i>Spe</i>I and <i>Bam</i>HI.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>24.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>0.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Spe</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Bam</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>HI</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested | ||
+ | pET-15b with <i>Xba</i>I and <i>Bam</i>HI and dephosphorylated with 1µL BAP.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pET-15b</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>87µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>10µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xba</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Bam</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>HI</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We extracted DNA | ||
+ | from the Blue gel.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We added 10 µL H<sub>2</sub>O | ||
+ | to dried <i>YJL068C</i> gene and pET-15b.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We ligated them | ||
+ | and transformed it into <i>E.coli </i>cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>25th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We cultured them | ||
+ | furthermore.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>26th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We checked the | ||
+ | insertion of DNA by colony cracking.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We cultured each | ||
+ | transformant in LB liquid medium with ampicillin.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We added 20ul of | ||
+ | IPTG to them and incubated.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Cell free extract | ||
+ | was prepared and applied to SDS-PAGE to detect <i>YJL068C </i>protein.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>29th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Cell free extract | ||
+ | was prepared with FastBreak Cell Lysis Reagent and applied to SDS-PAGE.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>30th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We performed PCR to | ||
+ | amplify <i>YJL068C</i>.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>(Annealing temperature: | ||
+ | 55°C)</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>31th August</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We purified the <i>YJL068C</i> | ||
+ | (performed PCR at 8/30).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested <i>YJL068C</i> | ||
+ | with <i>Ban</i>II to check whether PCR succeeded or not.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We ligated <i>YJL068C</i> | ||
+ | and pSB1C3.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We digested <i>YJL068C</i> | ||
+ | with <i>Xho</i>I and <i>Nde</i>I for ligation.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We purified | ||
+ | pET-15b and digested with <i>Xho</i>I and <i>Nde</i>I.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>24µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Nde</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pET-15b</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>33µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>4µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Nde</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>DNA samples were | ||
+ | applied to 1% Blue gel electrophoresis and then purified.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We added 10 µL H<sub>2</sub>O | ||
+ | to dried <i>YJL068C</i> gene and pET-15b.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We ligated them | ||
+ | and transformed it into <i>E.coli </i>cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 1<sup>st</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Picked 32 colonies | ||
+ | of <i>YJL068C</i> transformants.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 4th</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Digested <i>YJL068C</i> | ||
+ | and pET-15b overnight.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Applied pET-15b | ||
+ | and <i>YJL068C</i> to the blue gel electrophoresis.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Isolated and purified | ||
+ | it.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 5<sup>th</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Purified PCR | ||
+ | products and dissolved 24ul of H<sub>2</sub>O.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Purified pET-15b.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Digested <i>YJL068C</i> | ||
+ | and pET-15b with <i>Xho</i>I and <i>Nde</i>I overnight.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>24µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Nde</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pET-15b</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>33µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>4µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Nde</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <span lang=EN-US style='font-size:11.0pt;font-family:"Century","serif"'><br | ||
+ | clear=all style='page-break-before:always'> | ||
+ | </span> | ||
+ | |||
+ | <p class=MsoNormal align=left style='text-align:left'><span lang=EN-US | ||
+ | style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 6th</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Applied <i>YJL068C</i> | ||
+ | and pET-15b(prepared on September 5th) to the blue gel electrophoresis.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Isolated and purified | ||
+ | it.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Ligation of | ||
+ | pET-15b with <i>YJL</i> <i>068C</i> (prep).</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=114 valign=top style='width:85.45pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>10µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=114 valign=top style='width:85.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pET-15b</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>10µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=114 valign=top style='width:85.45pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Ligation Mix</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>10µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Transformed <i>YJL068C</i> | ||
+ | into pET-15b <i>E. coli</i> cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Purified PCR | ||
+ | products and pET-15b were dissolved in 24.5ul of H<sub>2</sub>O.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'> and pET-15b were digested with <i>Xho</i>I | ||
+ | and <i>Nde</i>I(overnight).</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>24.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>0.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Nde</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pET-15b</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>24.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>0.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=90 valign=top style='width:67.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Nde</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Applied YJL<i>068C</i> | ||
+ | and pET-15b(prepared on September 5th) to the blue gel electrophoresis.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Isolated and | ||
+ | purified it.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Ligation pET-15b | ||
+ | and <i>YJL068C</i> (prep).</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Transformed YJL<i>068C</i> | ||
+ | into pET-15b E. coli cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 7<sup>th</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Picked 48 colonies | ||
+ | of <i>YJL068C </i>transformants.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Checked the | ||
+ | colonies by colony cracking.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Picked up the | ||
+ | appropriate colonies and cultured in 3ml LB medium with ampicillin.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <span lang=EN-US style='font-size:11.0pt;font-family:"Century","serif"'><br | ||
+ | clear=all style='page-break-before:always'> | ||
+ | </span> | ||
+ | |||
+ | <p class=MsoNormal align=left style='text-align:left'><span lang=EN-US | ||
+ | style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 8<sup>th</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Plasmid DNA was | ||
+ | prepared by miniprep and digested with <i>Xho</i>I/<i>Nde</i>I.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>(1)</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Plasmid DNA</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>15.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>0.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Nde</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>(2)</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Bsi</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>WI</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Buffer</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Plasmid DNA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>17µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Electrophoresed in | ||
+ | 1% agarose gel.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Not succeeded.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 15<sup>th</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Transformed <i>YJL068C</i> | ||
+ | into pET-15b <i>E. coli</i> cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 16<sup>th</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Picked 48 colonies | ||
+ | of <i>YJL068C </i>transformants.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 17th</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Checked the | ||
+ | colonies by colony cracking.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Picked up the | ||
+ | colonies and cultured in 3ml LB medium with ampicillin.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 18<sup>th</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Miniprepped | ||
+ | plasmid DNA(<i>YJL068C</i> into pET-15b) and digested it.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Electrophoresed in | ||
+ | 1% agarose gel.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Not succeeded.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <span lang=EN-US style='font-size:11.0pt;font-family:"Century","serif"'><br | ||
+ | clear=all style='page-break-before:always'> | ||
+ | </span> | ||
+ | |||
+ | <p class=MsoNormal align=left style='text-align:left'><span lang=EN-US | ||
+ | style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 19<sup>th</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'> and pET-15b were digested with <i>Xho</i>I | ||
+ | and <i>Blp</i>I.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>YJL068C</span></i></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>24.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>NEB Buffer 4</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>0.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Blp</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>pET-15b</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>24.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>NEB Buffer 4</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>3µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>BSA</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>0.5µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=119 valign=top style='width:89.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Blp</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=57 valign=top style='width:42.4pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Transformed <i>YJL068C</i> | ||
+ | into pET-15b <i>E. coli</i> cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 20<sup>th</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Picked 64 colonies | ||
+ | of YJL068C transformants.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 21st</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Checked the | ||
+ | colonies by colony cracking.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Picked up the | ||
+ | appropriate colonies and cultured in the LB in 3ml medium with ampicillin.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
+ | |||
+ | <p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>September 22<sup>nd</sup></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Miniprepped | ||
+ | plasmid DNA(<i>YJL068C</i> into pET-15b) and digested it.</span></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Plasmid DNA</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>16µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Xho</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><i><span lang=EN-US style='font-size:11.0pt'>Blp</span></i><span | ||
+ | lang=EN-US style='font-size:11.0pt'>I</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>1µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=120 valign=top style='width:89.9pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>NEB Buffer 4</span></p> | ||
+ | </td> | ||
+ | <td width=56 valign=top style='width:42.25pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0mm 5.4pt 0mm 5.4pt'> | ||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>2µL</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Electrophoresed in | ||
+ | 1% agarose gel.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>We could not get | ||
+ | any <i>YJL068C</i> into pET-15b <i>E. coli</i> cells.</span></p> | ||
+ | |||
+ | <p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'> </span></p> | ||
</div> | </div> | ||
+ | |||
+ | |||
</div> | </div> |
Latest revision as of 08:46, 27 September 2013
YJL068C
13th August
We designed the PCR primers to amplify YJL068C gene.
19th August
1. Make Primer mix
We added 102.2 µL and 100.3 µL H2O to the forward and reverse primers, respectively, to make 100 pmol/µL solutions.
We obtained 10 µL of each primer solution and added 80 µL H2O
to make the 10 pmol/µL primer mix.
2. PCR
We performed PCR to amplify the YJL068C gene.
Buffer |
50µL |
dNTP |
20µL |
Primer mix |
1µL |
Genome DNA |
0.5µL |
KOD-FX |
2µL |
H2O |
26.5µL |
(Annealing : 52°C)
We detected two DNA bands around 1400bp and 3000bp.
(The correct band is 1400bp.)
We changed annealing temperature to 54°C and 57°C and tried PCR again.
We detected a correct size DNA band around 1400bp.
The clear DNA band was amplified at 54˚C (annealing temperature).
We performed PCR to amplify YJL068C gene at 54°C of annealing temp (total volume was 500 µL).
20th August
1
We performed PCR to check whether the primers can work or not.
(Annealing temperature: 55°C)
We detected a DNA band of the correct size around 900bp.
2
We purified the YJL068C gene (PCR was performed on 8/19).
We digested YJL068C with BanII to check whether PCR succeeded or not.
We ligated YJL068C and pSB1C3.
YJL068C |
24µL |
M Buffer |
3µL |
BSA |
1µL |
XbaI |
1µL |
SpeI |
1µL |
YJL068C |
6µL |
H Buff. |
2µL |
H2O |
11µL |
BanII |
1µL |
We digested pSB1C3 with XbaI and SpeI at 37°C for 90 minutes.
pSB1C3 |
49µL |
M Buffer |
5.78µL |
BSA |
1µL |
XbaI |
1µL |
SpeI |
1µL |
We added 1µL BAP to dephosphorylate the pcb1c3 gene.
We electrophoresed the YJL068C gene and pSB1C3 (digested for ligation) in 1% Blue gel.
We saw the clear band of YJL068C, and not so clear band of pSB1C3.
They were extracted from the Blue gel.
We prepared the spare the spare in case of failure (made stocks of purified YJL068C and pSB1C3).
We purified YJL068C and pSB1C3 (these were in one tube) and ligated them.
We transformed it all (20 µL) into E.coli cells.
21th August
1
We checked the YJL068C gene (digested with Ban2 8/20) by electrophoresing.
We could see correct band (succeeded PCR).
2
We checked of the insert by colony cracking (transformed 8/20).
We couldn’t see clear bands, but cultured furthermore.
3.
We made the spare in case of failure (prepared 8/20).
We purified the YJL068C gene (digested 8/20).
We digested pSB1C3 with XbaI and SpeI (90 minutes at 37°C), and added 1µL BAP to it.
pSB1C3 |
96µL |
Buffer |
11µL |
BSA |
1µL |
XbaI |
1µL |
SpeI |
1µL |
We electrophoresed pSB1C3 it in 1% Blue gel.
It was extracted from the Blue gel.
YJL068C |
5µL |
pSB1C3 |
5µL |
Ligation Mix |
10µL |
We added 5 µL H2O to dried YJL068C gene and pSB1C3 each.
We ligated them and transformed it into E.coli cells.
22th August
1.
We did miniprep to purify DNA.
We digested YJL068C in pSB1C3 with PstI and EcoRI, and incubated at 37°C for 90 minutes.
Plasmid DNA |
10µL |
H Buffer |
2µL |
H2O |
6µL |
EcoRI |
1µL |
PstI |
1µL |
We applied them to 1% agarose gel electrophoresis and checked them.
5 samples showed the correct size.
2.
We digested pSB1C3 with BsiWI.
pSB1C3 |
10µL |
NEB Buffer 3 |
2µL |
BsiWI |
1µL |
H2O |
7µL |
23th August
1.
We applied YJL068C in pSB1C3 (prepared on 8/22) to 1% agarose gel electrophoresis.
We detected the correct size of DNA.
Each transformant was cultured in LB liquid medium with chloramphenicol.
Plasmid DNA was purified by miniprep.
24th August
We added 29.5µL of H2O to purified YJL068C.
We digested YJL068C with BanII and checked.
YJL068C |
5µL |
H Buff. |
2µL |
H2O |
12µL |
BanII |
1µL |
We detected the correct size of DNA band.
We digested YJL068C with SpeI and BamHI.
YJL068C |
24.5µL |
Buffer |
3µL |
BSA |
0.5µL |
SpeI |
1µL |
BamHI |
1µL |
We digested pET-15b with XbaI and BamHI and dephosphorylated with 1µL BAP.
pET-15b |
87µL |
Buffer |
10µL |
BSA |
1µL |
XbaI |
1µL |
BamHI |
1µL |
We extracted DNA from the Blue gel.
We added 10 µL H2O to dried YJL068C gene and pET-15b.
We ligated them and transformed it into E.coli cells.
25th August
We cultured them furthermore.
26th August
We checked the insertion of DNA by colony cracking.
We cultured each transformant in LB liquid medium with ampicillin.
We added 20ul of IPTG to them and incubated.
Cell free extract was prepared and applied to SDS-PAGE to detect YJL068C protein.
29th August
Cell free extract was prepared with FastBreak Cell Lysis Reagent and applied to SDS-PAGE.
30th August
We performed PCR to amplify YJL068C.
(Annealing temperature: 55°C)
31th August
We purified the YJL068C (performed PCR at 8/30).
We digested YJL068C with BanII to check whether PCR succeeded or not.
We ligated YJL068C and pSB1C3.
We digested YJL068C with XhoI and NdeI for ligation.
We purified pET-15b and digested with XhoI and NdeI.
YJL068C |
24µL |
Buffer |
3µL |
BSA |
1µL |
XhoI |
1µL |
NdeI |
1µL |
pET-15b |
33µL |
Buffer |
4µL |
BSA |
1µL |
XhoI |
1µL |
NdeI |
1µL |
DNA samples were applied to 1% Blue gel electrophoresis and then purified.
We added 10 µL H2O to dried YJL068C gene and pET-15b.
We ligated them and transformed it into E.coli cells.
September 1st
Picked 32 colonies of YJL068C transformants.
September 4th
Digested YJL068C and pET-15b overnight.
Applied pET-15b and YJL068C to the blue gel electrophoresis.
Isolated and purified it.
September 5th
Purified PCR products and dissolved 24ul of H2O.
Purified pET-15b.
Digested YJL068C and pET-15b with XhoI and NdeI overnight.
YJL068C |
24µL |
Buffer |
3µL |
BSA |
1µL |
XhoI |
1µL |
NdeI |
1µL |
pET-15b |
33µL |
Buffer |
4µL |
BSA |
1µL |
XhoI |
1µL |
NdeI |
1µL |
September 6th
1.
Applied YJL068C and pET-15b(prepared on September 5th) to the blue gel electrophoresis.
Isolated and purified it.
Ligation of pET-15b with YJL 068C (prep).
YJL068C |
10µL |
pET-15b |
10µL |
Ligation Mix |
10µL |
Transformed YJL068C into pET-15b E. coli cells.
2.
Purified PCR products and pET-15b were dissolved in 24.5ul of H2O.
YJL068C and pET-15b were digested with XhoI and NdeI(overnight).
YJL068C |
24.5µL |
Buffer |
3µL |
BSA |
0.5µL |
XhoI |
1µL |
NdeI |
1µL |
pET-15b |
24.5µL |
Buffer |
3µL |
BSA |
0.5µL |
XhoI |
1µL |
NdeI |
1µL |
Applied YJL068C and pET-15b(prepared on September 5th) to the blue gel electrophoresis.
Isolated and purified it.
Ligation pET-15b and YJL068C (prep).
Transformed YJL068C into pET-15b E. coli cells.
September 7th
Picked 48 colonies of YJL068C transformants.
Checked the colonies by colony cracking.
Picked up the appropriate colonies and cultured in 3ml LB medium with ampicillin.
September 8th
Plasmid DNA was prepared by miniprep and digested with XhoI/NdeI.
(1)
Plasmid DNA |
15.5µL |
Buffer |
2µL |
BSA |
0.5µL |
XhoI |
1µL |
NdeI |
1µL |
(2)
BsiWI |
1µL |
Buffer |
2µL |
Plasmid DNA |
17µL |
Electrophoresed in 1% agarose gel.
Not succeeded.
September 15th
Transformed YJL068C into pET-15b E. coli cells.
September 16th
Picked 48 colonies of YJL068C transformants.
September 17th
Checked the colonies by colony cracking.
Picked up the colonies and cultured in 3ml LB medium with ampicillin.
September 18th
Miniprepped plasmid DNA(YJL068C into pET-15b) and digested it.
Electrophoresed in 1% agarose gel.
Not succeeded.
September 19th
YJL068C and pET-15b were digested with XhoI and BlpI.
YJL068C |
24.5µL |
NEB Buffer 4 |
3µL |
BSA |
0.5µL |
XhoI |
1µL |
BlpI |
1µL |
pET-15b |
24.5µL |
NEB Buffer 4 |
3µL |
BSA |
0.5µL |
XhoI |
1µL |
BlpI |
1µL |
Transformed YJL068C into pET-15b E. coli cells.
September 20th
Picked 64 colonies of YJL068C transformants.
September 21st
Checked the colonies by colony cracking.
Picked up the appropriate colonies and cultured in the LB in 3ml medium with ampicillin.
September 22nd
Miniprepped plasmid DNA(YJL068C into pET-15b) and digested it.
Plasmid DNA |
16µL |
XhoI |
1µL |
BlpI |
1µL |
NEB Buffer 4 |
2µL |
Electrophoresed in 1% agarose gel.
We could not get any YJL068C into pET-15b E. coli cells.