Team:WHU-China/Notebook/Protocols
From 2011.igem.org
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#protologo | #protologo | ||
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</span></b></p> | </span></b></p> | ||
- | <p class=MsoListParagraph style='text- | + | <p class=MsoListParagraph style='text-indent:2.0cm'><b><span lang=EN-US |
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif";color:red'> </span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:21.3pt'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif";color:red'>Attention:</span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>[1]<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>The efficiency of SpeI is relatively low, | ||
+ | therefore the amount of DNA can be decreased. The efficiency of EcoR</span><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>Ⅰ</span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>and | ||
+ | Xba</span><span style='font-size:12.0pt;line-height:150%;font-family:宋体'>Ⅰ</span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>is | ||
+ | relatively high, therefore the enzymes can be diluted two-three fold.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>[2]<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>The type of buffer should be selected | ||
+ | according to the restriction enzymes. </span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>[3]<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>The amount of DNA should not exceed 4ug</span><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>;</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>[4]<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>The enzymes should be stored on the ice | ||
+ | all the time. The buffer should be used after being thawed and blended.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>[5]<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Quick operation is recommended.</span></p> | ||
+ | |||
+ | <a name="ge"> | ||
+ | <p class=MsoNormal align=left style='margin-left:-.2pt;text-align:left'><span | ||
+ | style='font-size:20.0pt;font-family:宋体'>Ⅵ</span><span lang=EN-US | ||
+ | style='font-size:20.0pt;font-family:"Times New Roman","serif"'>. Get Extraction</span></p></a> | ||
+ | |||
+ | <p class=MsoNormal style='line-height:150%'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Things | ||
+ | to do before starting:</span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.55pt;text-indent:0cm; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>• Dilute SPW Wash Buffer with absolute | ||
+ | ethanol</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.55pt;text-indent:0cm; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>• Set heating block or water bath to | ||
+ | 60°C</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>1.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Perform agarose gel/ethidium bromide | ||
+ | electrophoresis to fractionate DNA fragments. Any type or grade of agarose may | ||
+ | be used. However, it is strongly recommended that fresh TAE buffer or TBE | ||
+ | buffer be used as running buffer. Do not reuse running buffer as its pH will | ||
+ | increase and reduce yields.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>When adequate separation of bands has | ||
+ | occurred, carefully excise the DNA fragment of interest using a wide, clean, | ||
+ | sharp scalpel. Minimize the size of the gel slice by removing extra agarose.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Determine the appropriate volume of the | ||
+ | gel slice by weighing it in a clean 1.5 ml microcentrifuge tube. Assuming a | ||
+ | density of 1g/ml of gel, the volume of gel is derived as follows: a gel slice | ||
+ | of mass 0.3 g will have a volume of 0.3 ml. </span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Add an equal volume of Binding Buffer | ||
+ | (XP2). Incubate the mixture at 60°C for 7 min or until the gel has completely | ||
+ | melted. Mix by shaking or vortexing the tube in increments of 2-3 minutes.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:0cm;line-height: | ||
+ | 150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;font-family: | ||
+ | "Times New Roman","serif";color:red'>IMPORTANT: Monitor the pH of the | ||
+ | Gel/Binding Buffer mixture after the gel has completely dissolved. DNA yields | ||
+ | will significantly decrease when the pH > 8.0. If the color of the mixture | ||
+ | becomes orange or red, add 5 μl of 5M sodium acetate (pH 5.2) to bring the pH | ||
+ | down. After this adjustment, the color of the Gel/Binding Buffer mixture should | ||
+ | be light yellow.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>5.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Place a HiBind DNA Mini Column in a | ||
+ | provided 2 ml collection tube.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>6.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Add the DNA/agarose solution from step 3 | ||
+ | to the HiBind DNA Mini Column and centrifuge at 10, 000 x g for 1 min at room | ||
+ | temperature. Discard the flow-through liquid and place the HiBind DNA Mini | ||
+ | Column back into the same collection tube. </span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:0cm;line-height: | ||
+ | 150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;font-family: | ||
+ | "Times New Roman","serif";color:red'>Note: For volumes greater than 700 μl, | ||
+ | load the column and centrifuge successively, 700 μl at a time. Each HiBind DNA | ||
+ | Mini Column has a total capacity of 25 μg DNA. If the expected yield is larger, | ||
+ | divide the sample into the appropriate number of columns.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>7.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Add 300 μl of Binding Buffer (XP2) into | ||
+ | the HiBind DNA Mini Column. Centrifuge at 13,000 x g for 1 minute at room | ||
+ | temperature. Discard the flow-through liquid and place the HiBind DNA Mini | ||
+ | Column back into the same collection tube.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>8.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Add 700 μl of SPW Wash Buffer (diluted | ||
+ | with absolute ethanol) to the HiBind DNA Mini Column. Centrifuge at 13,000 x g | ||
+ | for 1 min at room temp to wash the HiBind DNA Mini Column. Discard the flow-through | ||
+ | liquid and place the HiBind DNA Mini Column back into the same collection tube.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:0cm;line-height: | ||
+ | 150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;font-family: | ||
+ | "Times New Roman","serif";color:red'>IMPORTANT: SPW Wash Buffer must be diluted | ||
+ | with absolute ethanol before use.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:0cm;line-height: | ||
+ | 150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;font-family: | ||
+ | "Times New Roman","serif";color:red'>Refer to label for instructions. If | ||
+ | refrigerated, SPW Wash Buffer must be brought to room temperature before use.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>9.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'> OPTIONAL: Repeat steps 7 with | ||
+ | another 700 μl of SPW Wash Buffer.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>10.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Perform the second wash step for any | ||
+ | salt sensitive downstream applications</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>11.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Centrifuge the empty HiBind DNA Mini | ||
+ | Column for 2 min at maximal speed ( ≥13,000 x g ) to dry the column matrix. Do | ||
+ | not skip this step, it is critical for the removal of ethanol from the HiBind | ||
+ | DNA column.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>12.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Place the HiBind DNA Mini Column into a | ||
+ | clean 1.5 ml microcentrifuge tube.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>13.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Depending on desire concentration of the | ||
+ | final product, add 30-50 μl Elution Buffer (10 mM Tris-HCl, pH 8.5) or water | ||
+ | directly onto the column matrix and incubate at room temperature for 2 minutes. | ||
+ | Centrifuge for 1 min at maximum speed (≥13,000 x g) to elute DNA.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>14.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>This represents approximately 70% of | ||
+ | bound DNA. An optional second elution will yield any residual DNA, though at a | ||
+ | lower concentration.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:0cm;line-height: | ||
+ | 150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;font-family: | ||
+ | "Times New Roman","serif";color:red'>NOTE: The efficiency of eluting DNA from | ||
+ | the HiBind DNA Mini Column is dependent of pH. If eluting DNA with deionized | ||
+ | water, make sure that the pH is around 8.5.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.3pt;text-indent:-21.3pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>15.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>Yield and quality of DNA: Determine the | ||
+ | absorbance of an appropriate dilution of the sample at 260 nm and then at | ||
+ | 280nm.</span></p> | ||
+ | |||
+ | <a name="iod"> | ||
+ | <p class=MsoNormal align=left style='margin-left:-.2pt;text-align:left'><span | ||
+ | style='font-size:20.0pt;font-family:宋体'>Ⅶ</span><span lang=EN-US | ||
+ | style='font-size:20.0pt;font-family:"Times New Roman","serif"'>. Identification | ||
+ | of DNA by Electrophoresis</span></p></a> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:24.1pt;line-height:150%'><b><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Marker: | ||
+ | </span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph align=center style='text-align:center;text-indent: | ||
+ | 24.0pt;line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height: | ||
+ | 150%;font-family:"Times New Roman","serif"'><img border=0 width=224 height=332 | ||
+ | id="图片 1" src="/wiki/images/f/f1/Whu-Proto2.jpg" alt="说明: Marker-5000"></span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'> | ||
+ | <img border=0 width=113 height=334 id="图片 2" src="/wiki/images/8/83/Whu-Proto3.jpg" | ||
+ | alt="说明: Marker-2000"></span></p> | ||
+ | |||
+ | <p class=MsoListParagraph align=center style='text-align:center;text-indent: | ||
+ | 24.1pt;line-height:150%'><b><span lang=EN-US style='font-size:12.0pt; | ||
+ | line-height:150%;font-family:"Times New Roman","serif"'>λDNA/Hind</span></b><b><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>Ⅲ</span></b><b><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>+EcoR</span></b><b><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>Ⅰ</span></b><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'> | ||
+ | <b>DL 2000</b></span></p> | ||
+ | |||
+ | <a name="li"> | ||
+ | <p class=MsoNormal align=left style='text-align:left'><span style='font-size: | ||
+ | 20.0pt;font-family:宋体'>Ⅷ</span><span lang=EN-US style='font-size:20.0pt; | ||
+ | font-family:"Times New Roman","serif"'>. Ligation</span></p></a> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:2.0cm'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>System</span></b><b><span | ||
+ | style='font-size:12.0pt;font-family:宋体'>:</span></b><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>30ul</span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:0cm'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'> </span></b></p> | ||
+ | |||
+ | <table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=444 | ||
+ | style='width:333.1pt;margin-left:97.55pt;border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=246 valign=top style='width:184.3pt;border:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>T4 Ligase</span></p> | ||
+ | </td> | ||
+ | <td width=198 valign=top style='width:148.8pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>1ul</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=246 valign=top style='width:184.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>10XBuffer</span></p> | ||
+ | </td> | ||
+ | <td width=198 valign=top style='width:148.8pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>3ul</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=246 valign=top style='width:184.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>DNA fragment</span></p> | ||
+ | </td> | ||
+ | <td width=198 valign=top style='width:148.8pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>According to | ||
+ | the concentration of the DNA fragment</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=246 valign=top style='width:184.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Vector</span></p> | ||
+ | </td> | ||
+ | <td width=198 valign=top style='width:148.8pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>According to | ||
+ | the concentration of the vector</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=246 valign=top style='width:184.3pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>ddH<sub>2</sub>O</span></p> | ||
+ | </td> | ||
+ | <td width=198 valign=top style='width:148.8pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoListParagraph style='text-indent:0cm'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>To meet 30ul</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:2.0cm'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'> </span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:2.0cm'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Incubate the | ||
+ | reaction mixtures at 37</span></b><b><span style='font-size:12.0pt;font-family: | ||
+ | 宋体'>℃</span></b><b><span lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'> | ||
+ | for 2-3 hours.</span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.0pt;text-indent:0cm'><span | ||
+ | lang=EN-US style='font-family:"Times New Roman","serif"'> </span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='line-height:150%'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"; | ||
+ | color:red'>Attention</span></b><b><span style='font-size:12.0pt;line-height: | ||
+ | 150%;font-family:宋体;color:red'>:</span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>[1]<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>The ratio of DNA fragments to vector | ||
+ | should be 3-5:1</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>[2]<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>The ligation time should not be too | ||
+ | long.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.0pt;text-indent:0cm;line-height: | ||
+ | 150%'><span lang=EN-US style='font-family:"Times New Roman","serif"'>[3]<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>If | ||
+ | there are several DNA fragments to be ligated to the same vector, the sequence | ||
+ | should be optimized according to actual conditions.</span><span lang=EN-US | ||
+ | style='font-family:"Times New Roman","serif"'> </span></p> | ||
+ | |||
+ | <a name="pot"> | ||
+ | <p class=MsoNormal align=left style='text-align:left'><span style='font-size: | ||
+ | 20.0pt;font-family:宋体'>Ⅸ</span><span lang=EN-US style='font-size:20.0pt; | ||
+ | font-family:"Times New Roman","serif"'>. PCR of the colony</span></p></a> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:129.75pt'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>System</span></b><b><span | ||
+ | style='font-size:12.0pt;font-family:宋体'>:</span></b><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>20ul</span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Taq | ||
+ | 0.5ul</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Primer | ||
+ | 1 | ||
+ | 0.6ul</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Primer | ||
+ | 2 | ||
+ | 0.6ul</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>dNTP | ||
+ | 0.8ul</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Template | ||
+ | 1ul</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>DMSO | ||
+ | 1.2ul</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>10 X | ||
+ | Buffer | ||
+ | 2ul</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>ddH2O | ||
+ | 13.3ul</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:129.75pt'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Reaction | ||
+ | condition</span></b><b><span style='font-size:12.0pt;font-family:宋体'>:</span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>94</span><span | ||
+ | style='font-size:12.0pt;font-family:宋体'>℃</span><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'> | ||
+ | 5min</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>94</span><span | ||
+ | style='font-size:12.0pt;font-family:宋体'>℃</span><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'> | ||
+ | 30sec</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>55-68</span><span | ||
+ | style='font-size:12.0pt;font-family:宋体'>℃</span><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'> | ||
+ | 30sec</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>72</span><span | ||
+ | style='font-size:12.0pt;font-family:宋体'>℃</span><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'> | ||
+ | 1min</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>72</span><span | ||
+ | style='font-size:12.0pt;font-family:宋体'>℃</span><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'> | ||
+ | 7min</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='text-indent:184.3pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>28cycles</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:21.0pt;text-indent:0cm'><span | ||
+ | lang=EN-US style='font-family:"Times New Roman","serif"'> </span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='line-height:150%'><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"; | ||
+ | color:red'>Attention</span></b><b><span style='font-size:12.0pt;line-height: | ||
+ | 150%;font-family:宋体;color:red'>:</span></b></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt'><span | ||
+ | lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>[1]<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span><span | ||
+ | lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Pick | ||
+ | ten single colonies for each cloning.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt'><span | ||
+ | lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>[2]<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span><span | ||
+ | lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>The | ||
+ | PCR mix should be a little more than needed, in case of the loss during | ||
+ | operation.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt'><span | ||
+ | lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>[3]<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span><span | ||
+ | lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>DMSO | ||
+ | should be added into each PCR tube separately.</span></p> | ||
+ | |||
+ | <p class=MsoListParagraph style='margin-left:42.0pt;text-indent:-21.0pt'><span | ||
+ | lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>[4]<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span><span | ||
+ | lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>The | ||
+ | PCR mix should be blended.</span></p> | ||
+ | |||
+ | <a name="iods"> | ||
+ | <p class=MsoNormal><span style='font-size:20.0pt;font-family:宋体'>Ⅹ</span><span | ||
+ | lang=EN-US style='font-size:20.0pt;font-family:"Times New Roman","serif"'>.Identification | ||
+ | of DNA by Sequencing</span></p></a> | ||
+ | |||
+ | <p class=MsoNormal style='text-indent:42.0pt'><span lang=EN-US | ||
+ | style='font-size:12.0pt;font-family:"Times New Roman","serif"'>Send the | ||
+ | plasmids or the cell culture to BGI (A sequencing company in China) for | ||
+ | sequencing.</span></p> | ||
+ | |||
+ | <a name="ot"> | ||
+ | <p class=MsoNormal align=left style='margin-left:-.2pt;text-align:left'><span | ||
+ | lang=EN-US style='font-size:20.0pt;font-family:"Times New Roman","serif"'>XI.Others</span></p></a> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>1</span></b><b><span style='font-size: | ||
+ | 12.0pt;line-height:150%;font-family:宋体'>、</span></b><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>LB | ||
+ | medium components</span></b></p> | ||
+ | |||
+ | <table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=621 | ||
+ | style='width:466.1pt;border-collapse:collapse;border:none'> | ||
+ | <tr> | ||
+ | <td width=225 style='width:168.45pt;border:solid windowtext 1.0pt;padding: | ||
+ | 0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height: | ||
+ | 150%;font-family:"Times New Roman","serif"'>Volume</span></b></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.8pt;border:solid windowtext 1.0pt;border-left: | ||
+ | none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>100ml liquid medium</span></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.85pt;border:solid windowtext 1.0pt;border-left: | ||
+ | none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>100ml solid medium</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=225 style='width:168.45pt;border:solid windowtext 1.0pt;border-top: | ||
+ | none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height: | ||
+ | 150%;font-family:"Times New Roman","serif"'>Trptone</span></b></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.8pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>1g</span></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.85pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>1g</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=225 style='width:168.45pt;border:solid windowtext 1.0pt;border-top: | ||
+ | none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height: | ||
+ | 150%;font-family:"Times New Roman","serif"'>Yeast Extraction Powder</span></b></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.8pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>0.5g</span></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.85pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>0.5g</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=225 style='width:168.45pt;border:solid windowtext 1.0pt;border-top: | ||
+ | none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height: | ||
+ | 150%;font-family:"Times New Roman","serif"'>NaCl</span></b></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.8pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>1g</span></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.85pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>1g</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width=225 style='width:168.45pt;border:solid windowtext 1.0pt;border-top: | ||
+ | none;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height: | ||
+ | 150%;font-family:"Times New Roman","serif"'>Agar</span></b></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.8pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'> </span></p> | ||
+ | </td> | ||
+ | <td width=198 style='width:148.85pt;border-top:none;border-left:none; | ||
+ | border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal align=center style='margin-right:18.0pt;text-align:center; | ||
+ | line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>1.5g</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>2</span></b><b><span style='font-size: | ||
+ | 12.0pt;line-height:150%;font-family:宋体'>、</span></b><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Amp | ||
+ | mother solution</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | text-indent:24.0pt;line-height:150%'><span lang=EN-US style='font-size:12.0pt; | ||
+ | line-height:150%;font-family:"Times New Roman","serif"'>Preparation</span><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>:</span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Add | ||
+ | 100mg ampicillin into 1mlddH2O</span></p> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | text-indent:24.0pt;line-height:150%'><span lang=EN-US style='font-size:12.0pt; | ||
+ | line-height:150%;font-family:"Times New Roman","serif"'>Usage</span><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>:</span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Add | ||
+ | 50ulmother solution into 100ml medium</span></p> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>3</span></b><b><span style='font-size: | ||
+ | 12.0pt;line-height:150%;font-family:宋体'>、</span></b><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Kana | ||
+ | mother solution</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | text-indent:24.0pt;line-height:150%'><span lang=EN-US style='font-size:12.0pt; | ||
+ | line-height:150%;font-family:"Times New Roman","serif"'>Preparation</span><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>:</span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Add | ||
+ | 50mg kanamycin into 1mlddH2O</span></p> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | text-indent:24.0pt;line-height:150%'><span lang=EN-US style='font-size:12.0pt; | ||
+ | line-height:150%;font-family:"Times New Roman","serif"'>Usage</span><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>:</span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Add | ||
+ | 100ulmother solution into 100ml medium</span></p> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | line-height:150%'><b><span lang=EN-US style='font-size:12.0pt;line-height:150%; | ||
+ | font-family:"Times New Roman","serif"'>4</span></b><b><span style='font-size: | ||
+ | 12.0pt;line-height:150%;font-family:宋体'>、</span></b><b><span lang=EN-US | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Chl | ||
+ | mother solution</span></b></p> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | text-indent:24.0pt;line-height:150%'><span lang=EN-US style='font-size:12.0pt; | ||
+ | line-height:150%;font-family:"Times New Roman","serif"'>Preparation</span><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>:</span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Add | ||
+ | 50mg kanamycin into 1mlddH2O</span></p> | ||
+ | |||
+ | <p class=MsoNormal align=left style='margin-right:18.0pt;text-align:left; | ||
+ | text-indent:24.0pt;line-height:150%'><span lang=EN-US style='font-size:12.0pt; | ||
+ | line-height:150%;font-family:"Times New Roman","serif"'>Usage</span><span | ||
+ | style='font-size:12.0pt;line-height:150%;font-family:宋体'>:</span><span | ||
+ | lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>Add | ||
+ | 100ulmother solution into 100ml medium</span></p> | ||
+ | |||
+ | <p class=MsoNormal style='line-height:150%'><span lang=EN-US style='font-size: | ||
+ | 12.0pt;line-height:150%;font-family:"Times New Roman","serif"'> </span></p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div style="height:100px"></div> | ||
+ | </div> | ||
+ | |||
+ | </body> | ||
+ | </html> |
Revision as of 03:37, 23 September 2011