基因选殖的策略PPT课件[通用]

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,按一下以編輯母片標題樣式,按一下以編輯母片,第二層,第三層,第四層,第五層,*,*,*,基因選殖的策略,原核生物-細菌,萃取chromosome DNA,Partial digestion DNA(選一種限制酶),Vector(去磷酸根)(降低self-ligation),Ligation,trnasformation,藍白篩選,活性篩選,真核生物?,M-Marker:,DNA/HindIII,1-,Streptomyces,PMS502 chromosome,2-1/BamHI(,m,g DNA/0.18 unit),3-1/BamHI(,m,g DNA/0.06 unit),4-1/BamHI(,m,g DNA/0.02 unit),5-1/BamHI(,m,g DNA/0.007 unit),23.1,9.4,6.6,4.4,2.3,2.0,M,1 2 3 4 5,ATAACAATTTCACAC,AGGAA,ACAGCT,ATG,ACC,ATG,ATTAC,GAATTC,GAGCTC,GGTACC,Ligation,Transformation,Picking white colonies,選殖基因的定序,Clone確認,定序primer的選擇,Vector上的forward reverse序列,Ex:,pUC,18,第二次定序自行設計primer,序列的組合(正股 反股),Determining DNA sequences,1975 Frederick Sanger dideoxynucleoside triphosphates(ddNTPs),定序完成後的工作,上網比對序列blast確認所clone的基因,預測ORF(open reading frame),DNA-STAR,VECTOR NTI 9.0,ORF的確認,Ribosome binding site(RBS-6 -12 GA rich),蛋白的分子量與基因長度相關性,TATA box(-35 -10),設計primers subclone,expression,Expression vector 的選擇(pET,pQE,pGEX4T),基因產物的純化,設計Primers PCR基因產物前的分析工作,解讀限制酶圖譜,Primer序列加上限制酶切位,選擇哪種限制酶切位,Multi-cloning site有的切位,轉錄或轉譯起始位置的考量(與載體有關),primer設計時要注意序列 in frame,實做練習,ATAACAATTTCACAC,AGGAA,ACAGCT,ATG,ACC,ATG,ATTAC,GAATTC,GAGCTC,GGTACC,CGGAGCCGGGGAGCACATTGGCGGCACCGTGAGAGCGCTCTCAAAGGTCCCGCGTCCCCC 660,GCTCCCCGCTCCC,GAGAGG,CCACCC,ATG,AGTGATTCCTCCGGCACCCCCAGACCCCGTTC 720,M S D S S G T P R P R S,CCACTCCCGCCCCCGCTCCCGCTCCGTGCGCCGCGCCCTCATGGCGGCCGTCGCGACGTT 780,H S R P R S R S V R R A L M A A V A T F,CGGCCTCGCCGCGGCCGTCGCCACCGCGGCCACCGGCCCCGCGGACGCCTCCGCCCCGGC 840,G L A A A V A T A A T G P A D A S A P A,GCCGCCCTCCGGCTGGTCGCAGGTGTTCCTCGACGACTTCGACGGCGCGGCCGGAAGCAG 900,P P S G W S Q V F L D D F D G A A G S S,CGTCAACACCGCCAACTGGCAGTTCGACACCGGCACTTCGTACCCCGGCGGGGCAGGCAA 960,V N T A N W Q F D T G T S Y P G G A G N,CTGGGGCACTGGCGAGGTCGAGTCGATGACGTCGAGCACCAGCAATGTCTCCCTCGACGG 1020,W G T G E V E S M T S S T S N V S L D G,CAACGGCGATCTGCTCATCACCCCGCGCCGCGACGCGTCCGGCAACTGGACCTCCGGCCG 1080,N G D L L I T P R R D A S G N W T S G R,GATCGAGACCACCCGCACCGACTTCCAGCCCCCGGCCGGCGGCAAGCTGCGCGTCGAGGC 1140,I E T T R T D F Q P P A G G K L R V E A,GCGCCTGCAGATGCCGAACGTGACCGGGGACGCGGCCGCCGGGTACTGGCCCGCGTTCTG 1200,R L Q M P N V T G D A A A G Y W P A F W,GATGCTGGGCGCGCCCTTCCGCGGCAACTACCAGAACTGGCCCGGCGTCGGCGAGCTGGA 1260,M L G A P F R G N Y Q N W P G V G E L D,CATCATGGAGAACGTCCAGGGCCTGAACAAGACCTGGGCCACCATGCACTGCGGGACCAG 1320,I M E N V Q G L N K T W A T M H C G T S,CCCCGGCGGGCCCTGCAACGAGACCAGCGGCATCGGCAATTTGACCGCCTGCCCGAACAC 1380,P G G P C N E T S G I G N L T A C P N T,CACCTGCCATTCGGGCTTGCACACCTACACCATGGAGTGGGACCGCTCGGTCAGCCCCGA 1440,T C H S G L H T Y T M E W D R S V S P E,GGCGATCCGCTTCTCCGTCGACGGCGTCACCTACCAGACGGTCACGGCGAATCATATGGA 1500,A I R F S V D G V T Y Q T V T A N H M D,CGCGGTCACCTGGACCAACGCCACGAACCACGGTTTCTTCGTTATCCTGAACGTGGCGAT 1560,A V T W T N A T N H G F F V I L N V A M,GGGCGGCGGCTTCCCGGGTGCCTTCGGCGGCGGCCCGACCGGCGCCACCGAGCCCGGCCA 1620,G G G F P G A F G G G P T G A T E P G H,CCCCATGGTCGTCGACTACGTCCAGGTGCTCCAGTCCTCGGGCGGCGGGGGCGGCGGGGG 1680,P M V V D Y V Q V L Q S S,G G G G G G G,CGGGGGCACCACTCCCCCGCCGACCGGCGACCGGGACGCGTACGGTCAGATCCAGGCGGA 1740,G G,T T P P P T G,D R D A Y G Q I Q A E,GTCGTACGACGGCCAGTCCGGTGTCGCCACCGAGACCACCACGGACACCGGCGGCGGACA 1800,S Y D G Q S G V A T E T T T D T G G G Q,GGACATGGGCTACCTCGCCAATGGCGACTGGGCGCTGTACAAGGGCGTGAACTTCGGCTC 1860,D M G Y L A N G D W A L Y K G V N F G S,GACACCGGCCACCCAGTTCTACGGTCGGGTGGCGAGCGGCGCGGGCGGCGGGGTCAGCGG 1920,T P A T Q F Y G R V A S G A G G G V S G,CCTGGTGGAGGTGCGGCTGGACAGCCGTACGAACGCGCCGATCGGGAGCTTCGCGGTGGG 1980,L V E V R L D S R T N A P I G S F A V G,CGACACCGGGGGCTGGCAGTCCTGGCGGACGGTTCCGGCCAACATCGGGTCCGTCACCGG 2040,D T G G W Q S W R T V P A N I G S V T G,CACGCACGATGTGTACCTCACGTTCAGCAGCGGGCAGCCGGCCGACTTCGTGAACGTCAA 2100,T H D V Y L T F S S G Q P A D F V N V N,CTGGTTCGACTTCGGGCAC,TGA,TCCGCCGCGGGTACCGGGGTAGCGTTCACAGCATGTCG 2160,W F D F G H *,