[醫(yī)學(xué)]in-fusion克隆技術(shù)介紹20xxnew(編輯修改稿)
2025-02-15 10:16 本頁面
【文章內(nèi)容簡介】 signalCD101Fc ，并將其轉(zhuǎn)染 COS細(xì)胞檢測表達(dá)情況。實(shí)驗(yàn)流程1. 設(shè)計(jì)引物；2. 擴(kuò)增目的片段及載體線性化；3. 建立 InFusion連接反應(yīng)體系 。?InFusion174。應(yīng)用實(shí)例 多片段連接4/20/2022InFusion克隆技術(shù)介紹20 引物設(shè)計(jì)并擴(kuò)增目的片段 IL2 Signal, CD101和 Murine IgG3 IL2 Signal with Overlaps to NcoIDigested Vector and CD101, 88bp PCR ProductSense (NcoI underlined)5′TTCAAATCCACCATGGATAGAATGCAATTGTTG3′Antisense5′CTGTTACTTCTCTCTGAGAATTCGTAACCAAAGCCAAAGACAAAGCAATCA3′CD101, 2799bp PCR ProductSense5′CAGAGAGAAGTAACAGTTCAGAAA3′Antisense5′GGCCGAGGAGCAGATCCTGGAA3′Murine IgG3 with Overlaps to CD101 and SalIDigested Vector, 771bp PCR ProductSense5′ATCTGCTCCTCGGCCCCTAGAATACCCAAGCCCAGTACC3′Antisense (SalI underlined)5′AGTAACGTTAGTCGACTCAGTGTCTTGTAAGACCCGAGGA3′?InFusion174。應(yīng)用實(shí)例 多片段連接4/20/2022InFusion克隆技術(shù)介紹21 建立 InFusion連接反應(yīng)體系（ 10 μl ）IL2 Signal CD101 Domain Murine IgG3 Fc Tail Vector Total Transformants ErrorFree/ Sequence (No.)88 bp, ng 2799 bp, ng 771 bp, ng 11,365 bp, 100 ng 690 1/2結(jié)論：克隆陽性率為 50%。轉(zhuǎn)染 COS 細(xì)胞后，融合蛋白 IL2 signal CD101Fc表達(dá)情況良好。?InFusion174。應(yīng)用實(shí)例4/20/2022InFusion克隆技術(shù)介紹22 1 ?多片段連接實(shí)驗(yàn)2 ?定點(diǎn)突變實(shí)驗(yàn)?InFusion174。應(yīng)用實(shí)例 定點(diǎn)突變4/20/2022InFusion克隆技術(shù)介紹23 定點(diǎn)突變 完美實(shí)現(xiàn)單點(diǎn)多點(diǎn)突變實(shí)驗(yàn)流程1. 確定突變位點(diǎn)；2. 設(shè)計(jì)引物；3. 擴(kuò)增目的片段及載體線性化；4. 建立 InFusion連接反應(yīng)體系。?InFusion174。應(yīng)用實(shí)例 定點(diǎn)突變4/20/2022InFusion克隆技術(shù)介紹24 策略一5′ Sense Primer (Overlap with Vector Underlined, SpeI Site Bold) 5′GCTCGGATCCACTAGTCCAG
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