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[求助]
A noob intersted in SCI searching for help(about overlap PCR)
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With 14 rare codens in gene, the expression of AK404 maybe reduced. Thus, the rare codens were designed to be substituted by E.coli perferred codens Original AK404 gene being as templete, nine pairs of primers were designed to synthesis the new AK404 gene with E.coli perferred codens, as shown in Fig 1.Designed primers for sythesising new AK404 gene: Primer 1 with mutation sites 1 and 2; Primer 2 with mutation site 3; Primer 3 with mutation sites 4 and 5; Primer 4 with mutaion site 6; Primer 5 with mutaion site 7; Primer X with a mutaion orginal being an NcoI restriction site; Primer 6 with mutation sites 8,9 and 10; Primer 7 with mutaion site 11; Primer 8 with mutaion site 12 and 13; Primer 3’ with mutaion site 14. Firstly, using SOE PCR protocl, Primer5’, Primer1 and Primer 6 are designed to produce SubGene1A, as well as Primer3’, Primer7 and Primer 6 to SubGene1B. In principle, with SubGene1A coupling SubGene1B, the whole gene will be synthetized by means of overlap PCR. The rest can be done in the same manner  |
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The first step geting ready for the subsequent synthesis of SubGene1A and SubGene1B(with 10 cycles of Denature-Annealing-elongation): Line 1: AK404 primer5’ coupling mutaion 1 primer5’,named M01; Line 2: Mutation 6 primer5’ coupling mutation 7 primer3’, named M67; Line 3: Mutation 1 primer5’ coupling mutation 6 primer3’,named M16; Line 4: Mutation 7 primer5’ coupling AK404 primer3’, named M70. The localization of band in line 2 fitted with that have been anticipated, whereas the base number of band in line 4 showed was less than the that of orginal fragment. PCR condition: 94C 10min, (94C 1min, 50C 40s, 72C 40s)*10cycles, 72C 10min  |
3楼2011-11-09 10:24:08
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However, result is far from encouraging. The first step geting ready for the subsequent synthesis of SubGene1A and SubGene1B(with 5 cycles of Denature-Annealing-elongation): Line 1: AK404 primer5’ coupling mutaion 1 primer5’,named M01; Line 2: Mutation 6 primer5’ coupling mutation 7 primer3’, named M67; Line 3: Mutation 1 primer5’ coupling mutation 6 primer3’,named M16; Line 4: Mutation 7 primer5’ coupling AK404 primer3’, named M70. Firstly, because of extremely short distance between 5’ terminal AK404 gene and mutation 1 site, as well as mutation 7 site to mutation 6 site, the target gene is hard to distinct from primer dimer. Secondly, as the few cycles, the band seemed to be fuzzy and this may not be the problem (PCR condition: 94C 10min, (94C 1min, 50C 40s, 72C 40s)*5cycles, 72C 10min  |
2楼2011-11-09 10:18:12
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SubGene1A and SubGene1B: Line 3: Using the production M01 and M16 from step I(5 cycles) as templete and primers, the newly synthetzed gene was showen about 500bp long by the band in line 3, and it is conformed to that of SubGene1A anticipited. Line 4: Using the production M67 and M170 from step I(5 cycles) as templete and primers, the newly synthesized gene was showen about 300-400bp long by the band in line 4, and it is conformed to that of SubGene1B anticipited. PCR condition: 94C 10min, (94C 1min, 50C 40s, 72C 40s)*30 cycles, 72 10min  |
4楼2011-11-09 10:31:44
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SubGene1A and SubGene1B being templete and primers, new AK404 with four mutation sites was expected to be synthetized. Nevertheless, nothing but the fuzzy tailing has been showen in electrophoresis diagram. PCR condition:94C 5min, (94C 1min, 58C 70s, 72C 40s)*30 cycles, 72C 10min By the way ,SubGene1A and SubGene1B were gotten by Gel extraction  |
5楼2011-11-09 10:38:26