https://ilvwen.cn/Good Luck To You!https://ilvwen.cn/?id=26<ol class=" list-paddingleft-2" style="list-style-type: decimal;"><li><p>用XL10-Gold转化PJ449-2、PJ450-1、PJ98、PJ100和PJ147.</p></li><li><p>传代293T 1盘24孔板（明转染PJ448、PJ449和PJ450），1盘12孔板（保种）</p></li><li><p>转染PJ425、PJ427、PJ428、PJ429和PJ431，测试质粒是否有效。<br/></p></li></ol>Thu, 02 Jul 2026 10:40:43 +0800https://ilvwen.cn/?id=25<p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">冲洗管路</span></strong><span style=";font-family:宋体;font-size:16px">：</span><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">取干净的流式管，加入</span><span style="font-family:宋体">ddH</span></span>_{<span style=";font-family:宋体;font-size:16px;vertical-align:sub"><span style="font-family:宋体">2</span></span>}<span style=";font-family:宋体;font-size:16px">O，最大流速冲洗10 min。</span></p><p style="line-height:27px"><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">关闭液流：点击</span><span style="font-family:宋体">“strem”，液流关闭。</span></span></p><p style="line-height:27px"><span style=";font-family:宋体;font-size:16px">换上闭合喷嘴：取下流动喷嘴，换上闭合喷嘴。</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">冲洗流动池：</span></strong><span style=";font-family:宋体;font-size:16px">菜单</span><span style=";font-family:宋体;font-size:16px">-</span><span style=";font-family:宋体;font-size:16px">Cytometer→Cleaning Modes→Clean Flow Cell</span><span style=";font-family:宋体;font-size:16px">，</span><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">清洗</span><span style="font-family:宋体">2~3遍。</span></span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">启动关机程序：</span></strong><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">菜单</span><span style="font-family:宋体">—</span></span><span style=";font-family:宋体;font-size:16px">Cytometer→</span><span style=";font-family:宋体;font-size:16px">fluid shutdown，根据提示操作。</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">关闭激光：</span></strong><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">待流式软件关机程序结束，跳转激光软件，依次</span><span style="font-family:宋体">40-25-10-1递减功率，最后将三个激光全部关闭。在仪器上关闭激光电源。</span></span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">关</span></strong><strong><span style="font-family: 宋体;font-size: 16px">闭主设备：</span></strong><span style=";font-family:宋体;font-size:16px">关闭主设备电源。关闭流式软件。</span></p><p style="line-height:27px"><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">关闭电脑，关闭气泵，关闭</span><span style="font-family:宋体">UPS，关闭电源。</span></span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 21px">optional</span></strong><span style=";font-family:宋体;font-size:16px">：</span></p><p><strong><span style="font-family: 宋体;font-size: 16px"><span style="font-family:宋体">添加乙醇桶</span><span style="font-family:宋体">75%酒精：</span></span></strong><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">开盖，加入适量</span><span style="font-family:宋体">75%酒精。</span></span></p>Sun, 28 Jun 2026 16:58:41 +0800https://ilvwen.cn/?id=24<p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">硬件启动</span></strong><span style=";font-family:宋体;font-size:16px">：稳压电源</span><span style=";font-family:宋体;font-size:16px">-气泵-计算机(进桌面,</span><span style=";font-family:宋体;font-size:16px">BDIS</span><span style=";font-family:宋体;font-size:16px">#1)-刷卡-流式机-激光</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">激光</span></strong><span style=";font-family:宋体;font-size:16px">：软件</span><span style=";font-family:宋体;font-size:16px">-三个激光开启（355不开）-从小到大15，25，40w，最小化</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">流式</span></strong><span style=";font-family:宋体;font-size:16px">：软件</span><span style=";font-family:宋体;font-size:16px">-等待连接-对话框（use cst settings）</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">液流启动</span></strong><span style=";font-family:宋体;font-size:16px">：菜单</span><span style=";font-family:宋体;font-size:16px">-cytometer-fluidics setup</span></p><p style="line-height:27px"><span style=";font-family:宋体;font-size:16px">气路，液路更换至鞘液桶</span><span style=";font-family:宋体;font-size:16px">-黄钮下按拉出推车-气路连气路，液路连鞘液过滤器-推回去</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">闭合喷嘴在机器上</span></strong></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">去掉闭合喷嘴</span></strong><span style=";font-family:宋体;font-size:16px">：垂直挂在孔上</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">插入喷嘴</span></strong><span style=";font-family:宋体;font-size:16px">：红点朝上，插入后，旋转固定</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">打开液流</span></strong><span style=";font-family:宋体;font-size:16px">：点击</span><span style=";font-family:宋体;font-size:16px">stream，调节angel，将断点移至第四滴，将实际数值与上次数值靠近</span><span style=";font-family:宋体;font-size:16px">，</span><span style=";font-family:宋体;font-size:16px">固定。</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">调整</span></strong><strong><span style="font-family: 宋体;font-size: 16px">drop delay</span></strong><span style=";font-family:宋体;font-size:16px">:</span><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">左边文件树</span><span style="font-family:宋体">-</span></span><span style=";font-family:宋体;font-size:16px">20200603-accudrop，双击-continiue-accudrop beads-tube_004，关仓门</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">微球校准偏电</span></strong><span style=";font-family:宋体;font-size:16px">:4°拿微球（有颜色，无标识，流式管）-混匀-上机-流速调-点击load-</span><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">流速调至</span><span style="font-family:宋体">1000-3000 beads/s-</span></span><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">点击菜单</span>sort-new sort layout-add p</span><span style=";font-family:宋体;font-size:16px">1</span><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">（全部）</span>-sort-cancel-voltage-optical filter，调drop delay使得左一路</span><span style=";font-family:宋体;font-size:16px">9</span><span style=";font-family:宋体;font-size:16px">0%以上-auto delay-start run-exit-stop acuiring-unload-微球放回4°</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 21px">optional</span></strong><span style=";font-family:宋体;font-size:16px">：</span></p><p style="line-height:27px"><strong><span style="font-family: 宋体;font-size: 16px">中途添加鞘液：</span></strong><span style=";font-family:宋体;font-size:16px">菜单</span><span style=";font-family:宋体;font-size:16px">-cytometer-</span><span style=";font-family:宋体;font-size:16px">standby，仪器放气，打开鞘液桶，加鞘液，</span><span style=";font-family:宋体;font-size:16px">菜单</span><span style=";font-family:宋体;font-size:16px">-cytometer-</span><span style=";font-family:宋体;font-size:16px">connect，待软件</span><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">连接</span>-对话框（use cst settings）</span><span style=";font-family:宋体;font-size:16px">，</span><span style=";font-family:宋体;font-size:16px"><span style="font-family:宋体">点击</span><span style="font-family:宋体">stream，等待液流稳定，固定即可。</span></span></p><p style="line-height: 27px;"><strong><span style="font-family: 宋体;font-size: 16px">添加乙醇桶内酒精</span></strong><span style=";font-family:宋体;font-size:16px">：放气</span><span style=";font-family:宋体;font-size:16px">-打开-加液至线-关盖（黄色标签对着提手）</span></p>Sun, 28 Jun 2026 16:33:52 +0800https://ilvwen.cn/?id=23<ol class=" list-paddingleft-2" style="list-style-type: decimal;"><li><p>查找小鼠、绿猴的spcas9好用SgRNA</p></li><li><p>查找groupI、groupII的核酶<br/></p></li></ol>Sun, 21 Jun 2026 20:13:58 +0800https://ilvwen.cn/?id=22<p>466-7、8、11、13、15是TKO敲除，466-3的CMAH不确定是否敲除</p><p>3、8的状态较差，13和15的状态好些，所以可以分成两组（3、7，13）和（8，11，15）</p>Sat, 20 Jun 2026 16:17:12 +0800https://ilvwen.cn/?id=21<p>今日事</p><ol class=" list-paddingleft-2" style="list-style-type: decimal;"><li><p>流式分选465-466（已完成）</p><p>每个收回约5万5的细胞，转染效率在30%左右，每管分成三份，96孔上铺两份，剩余1份铺4个10 cm皿。多余细胞煮基因组。</p></li><li><p>PCR 465、466</p></li><li><p>转化PJ391</p></li></ol><p><br/></p>Fri, 29 May 2026 16:40:32 +0800https://ilvwen.cn/?id=20<p>今日事</p><ol class=" list-paddingleft-2" style="list-style-type: decimal;"><li><p>电转PFF（已完成）</p><p>转染编号465-466</p></li><li><p>统计3rd NGS数据</p></li><li><p>设计TSS-HI和Lonza 4D电转液配制方案</p></li><li><p>设计新质粒</p></li></ol>Thu, 28 May 2026 14:36:32 +0800https://ilvwen.cn/?id=19<p>生工订单2120910840：</p><p>445测序正确质粒：445-1、445-4、445-5</p><p>446测序正确质粒：445-3</p><p>已申请返样</p>Mon, 11 May 2026 10:43:00 +0800https://ilvwen.cn/?id=18<p>今日事</p><ol class=" list-paddingleft-2" style="list-style-type: decimal;"><li><p>对不同GFP变体进行规整。发现PJ395和PJ396有问题，需要重新构建。</p></li><li><p>复苏Bama31-7 P41管，1：2复苏两孔6孔板孔。</p></li></ol>Mon, 13 Apr 2026 13:55:34 +0800https://ilvwen.cn/?id=17<ol class=" list-paddingleft-2" style="list-style-type: decimal;"><li><p>用PBS当回收液，PCR结果优于DMEM。</p></li><li><p>细胞裂解液，每5000个细胞加5 uL，5000个细胞以下加5 uL。</p></li><li><p>5000个细胞及以上的粗裂解产物，第一轮反应1 uL当模板即可；如果是用EB稀释了1倍，<span style="text-wrap-mode: wrap;">第一轮反应2 uL当模板。</span></p></li><li><p><span style="text-wrap-mode: wrap;">500-5000个细胞<span style="text-wrap-mode: wrap;">需要2 uL作为第一轮反应模板<span style="text-wrap-mode: wrap;">；如果是用EB稀释了1倍，</span><span style="text-wrap-mode: wrap;">第一轮反应2 uL当模板</span>。</span></span></p></li></ol>Fri, 10 Apr 2026 10:53:51 +0800