| 查看: 1795 | 回复: 13 | ||
zhang8826857铁杆木虫 (著名写手)
|
[求助]
大家帮我保存个网页吧，我电脑上这个网页显示有问题，金币不够还有的哦
|
回复此楼» 本主题相关价值贴推荐，对您同样有帮助:
某些网页多次点击才能打开
已经有1人回复
- 怎么保存这个网页？
已经有5人回复
- ie浏览器其他网页可以进QQ空间和邮箱从QQ面板进不去~我郁闷
已经有9人回复
- 浏览器打开的网页显示不全？怎么办？
已经有3人回复
- 【求助】谁知道用制作网页的步骤？
已经有10人回复
- 求助笔记本无线网络显示连接好了且信号很好但打不开网页
已经有9人回复
- 【求助】用photoshop 如何保存eps格式的图片啊
已经有11人回复
- 【转帖】让网页中的PDF文件保存后再打开
已经有9人回复
zhouxiaohan
木虫 (正式写手)
- 应助: 2 (幼儿园)
- 金币: 1434.5
- 散金: 519
- 红花: 1
- 帖子: 810
- 在线: 189.5小时
- 虫号: 1210540
- 注册: 2011-02-23
- 专业: 医学光子学、光谱与光学成
2楼2011-12-06 09:42:54
3楼2011-12-06 09:43:22
mlanqiang
木虫之王 (文学泰斗)
蓝博士
- 应助: 3409 (副教授)
- 金币: 55278.8
- 散金: 5498
- 红花: 62
- 帖子: 73916
- 在线: 730.8小时
- 虫号: 302202
- 注册: 2006-12-02
- 性别: GG
- 专业: 胶体与界面化学
4楼2011-12-06 09:54:26
zhang8826857
铁杆木虫 (著名写手)
- 应助: 6 (幼儿园)
- 贵宾: 1.049
- 金币: 8847.6
- 散金: 606
- 红花: 72
- 沙发: 2
- 帖子: 2137
- 在线: 278.8小时
- 虫号: 1397074
- 注册: 2011-09-10
- 专业: 微生物生理与生物化学
5楼2011-12-06 10:59:29
zhang8826857
铁杆木虫 (著名写手)
- 应助: 6 (幼儿园)
- 贵宾: 1.049
- 金币: 8847.6
- 散金: 606
- 红花: 72
- 沙发: 2
- 帖子: 2137
- 在线: 278.8小时
- 虫号: 1397074
- 注册: 2011-09-10
- 专业: 微生物生理与生物化学
6楼2011-12-06 10:59:51
zhang8826857
铁杆木虫 (著名写手)
- 应助: 6 (幼儿园)
- 贵宾: 1.049
- 金币: 8847.6
- 散金: 606
- 红花: 72
- 沙发: 2
- 帖子: 2137
- 在线: 278.8小时
- 虫号: 1397074
- 注册: 2011-09-10
- 专业: 微生物生理与生物化学
7楼2011-12-06 11:00:02
西瓜
荣誉版主 (知名作家)
- MolEPI: 50
- 应助: 94 (初中生)
- 贵宾: 3.157
- 金币: 1849.6
- 散金: 11458
- 红花: 116
- 沙发: 21
- 帖子: 7553
- 在线: 1449.5小时
- 虫号: 271749
- 注册: 2006-08-13
- 性别: GG
- 专业: 细胞衰老
- 管辖: 分子生物
【答案】应助回帖
zhang8826857(金币+15): ^_^^_^^_^ 2011-12-06 11:29:19
zhang8826857: 回帖置顶 2011-12-06 11:42:58
zhang8826857: 回帖置顶 2011-12-06 11:42:58
|
Transposon-mediated Mutagenesis Step 1 - Amplify ORF from MG1655 1.0 ul genomic DNA (30ng/ul) 5.0 ul gene specific primer mix 4.0 ul 2.5 mM dNTP 5.0 ul 10x Turbo Pfu buffer 0.7 ul Turbo Pfu 34.3 ul sterile distilled H2O 50 ul 95°C 95°C 55°C 72°C 72°C 4°C 1min 15sec 15sec 4min 5min forever 25 cycles Step 2 - Transposition reaction 6 ul ORF PCR product (~100-200ng) 2 ul Tn (Transposon, 200ng = 0.2pM) 1 ul 10x Tn reaction buffer (Epicentre) 1 ul Tnase (Transposase; Epicentre) 10 ul Vortex, spin down 37°C for 2hr Add 1 ul stop solution, 10min 70°C Purify by passing through G50 column Step 3 – Transformation MG1655 cells harboring the pKD46 plasmid (Datsenko and Wanner, 2000, PNAS 97(12):6640-5) are induced with arabinose to activate expression of the lRed genes and prepared for electroporation. 1. Mix following on ice: 4.4 ul transposition reaction 40 ul MG1655 w/pKD46 electrocompetent cells 2. Transfer entire volume (~45ul) to chilled cubette (0.1cm gap), kept on ice 3. Electroporate with the following settings (for BioRad Pulse Controller) Low Range 200 High Range 500 Capacitance (uF): -25 Total Volts: 1.8kV 4. Resuspend cells with 1ml LB and transfer contents to a 48-well growth block 5. Outgrow at 37°C for 1hr 6. Spread entire outgrowth on a corresponding labeled LB + Amp(100ug/ml)/Kan (50ug/ml) plate in a hood and air dry. 7. Grow at 300 (to maintain pKD46). (1- 2 days) Step 4- Picking Day 1 1. Streak 2 colonies to single colonies on LB + Amp(100ug/ml)/Kan (50ug/ml) plates. These will be the first colonies to verify. 2. Pick 3 additional colonies into separate 96-well flat bottom plates containing 200ul Freezing media + Amp(100ug/ml)/Kan(50ug/ml) maintaining original well location. 3. Grow 300 overnight. Day 2: 1. Grow 1 colony from each streak plate in a 96-well block with each well containing 1ml Freezing media + Amp(100mg/ml)/Kan(50mg/ml). Grow 300 overnight. 2. Freeze overnights of the other 3 plates. Step 5– Verify mutation by Culture PCR 1. Prepare sample Mix culture thoroughly. Transfer 20ul of culture into a 96-well plate and dilute with 80ul H2O Mix thoroughly 2. PCR Reaction. 5 ul diluted culture 4 ul gene specific primer mix (same as in Step 1) 2.5 ul ExTaq premix 16 ul H2O 50 ul 95°C 94°C 55°C 72°C 72°C 4°C 5min 30sec 15 sec 4min 5min forever 30cycles 3. Run 7 ul on 1 % test gel in 0.5x TAE to check. 4. Analyze gel results; mutants will be ~1.2kb larger than original gene length. Step 6 - Confirm mutations by sequencing 1. EXOSAP clean up - Transfer 10ul of PCR product for all genes with mutant sized fragments into a separate PCR plate - Add 4ul ExoSAP-IT (USB) to each reaction - Spin briefly - React @ 37°C for 30min, then 15min@ 80°C 2. Sequence 1. Add a mix of the following to each ExoSAP reaction: 1ul of primer (KAN-2 FP-1 @ 10uM -Epicentre) 2ul Big Dye dilution Buffer (Promega) 3ul Big Dye 2. Reaction conditions: (10sec@96C; 5sec@50C; 4min@60C) for 25cycles. 3. Purify through a G50 column 4. Dry plate and run on sequencer 3. Make tube stocks of confirmed mutants Step 7 - Curing the temperature sensitive pKD46 plasmid 1. Streak confirmed mutants on a LB + Kan plate (from the above stock). Grow at 43° overnight (non-permissive temperature for pKD46 replication). 2. With a single colony inoculate the following: 1. Growth block well containing 1ml Freezing media + Kan(50ug/ml)/well . 2. LB+Kan agar in a 96 well plate 3. LB+Amp agar in a second 96-well plate 3. Grow overnight at 37°C. Cured cells will grow on Kan but not on Amp. 4. Confirm by PCR and sequencing as above. |
8楼2011-12-06 11:14:51
西瓜
荣誉版主 (知名作家)
- MolEPI: 50
- 应助: 94 (初中生)
- 贵宾: 3.157
- 金币: 1849.6
- 散金: 11458
- 红花: 116
- 沙发: 21
- 帖子: 7553
- 在线: 1449.5小时
- 虫号: 271749
- 注册: 2006-08-13
- 性别: GG
- 专业: 细胞衰老
- 管辖: 分子生物
9楼2011-12-06 11:17:29
西瓜
荣誉版主 (知名作家)
- MolEPI: 50
- 应助: 94 (初中生)
- 贵宾: 3.157
- 金币: 1849.6
- 散金: 11458
- 红花: 116
- 沙发: 21
- 帖子: 7553
- 在线: 1449.5小时
- 虫号: 271749
- 注册: 2006-08-13
- 性别: GG
- 专业: 细胞衰老
- 管辖: 分子生物
10楼2011-12-06 11:26:12
5