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zhang8826857铁杆木虫 (著名写手)
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[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
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zhouxiaohan
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2楼2011-12-06 09:42:54
3楼2011-12-06 09:43:22
mlanqiang
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4楼2011-12-06 09:54:26
zhang8826857
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5楼2011-12-06 10:59:29
zhang8826857
铁杆木虫 (著名写手)
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6楼2011-12-06 10:59:51
zhang8826857
铁杆木虫 (著名写手)
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7楼2011-12-06 11:00:02
西瓜
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【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
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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
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9楼2011-12-06 11:17:29
西瓜
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10楼2011-12-06 11:26:12
50