Phng php tch chit DNA v plasmid ca vi khun

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PHNG PHP TCH CHIT DNA V PLASMID CA VI KHUN

Phng php tch chit DNA v plasmid ca vi khun

CNG NGH SINH HC

Mi nghin cu v ng dng ca cng ngh gen u c bt u t vic thu nhn mt lng axit nucleic tinh sch. Mi quan tn u tin ca k thut tch chit axit nucleic l thu nhn c cc phn t ny trng thi nguyn vn, khng b phn hy bi tc nhn c hc, tc nhn ha hc v enzyme nh RNase, DNase. I. Phng php tch chit DNA vi khun DNA c kch thc ln, mi vic chit tch cn phi trnh mi tc nhn c hc, ho hc ti phn t DNA ny. Vic tch DNA t t bo vi khun c chia lm bn bc c bn: 1. Nui cy v thu sinh khi Ty tng loi vi khun m ngi nghin cu chun b mi trng v iu kin nui cy khc nhau nh nhit , pH, v iu kin dinh dng thch hp cho s pht trin ca vi khun. Tch t bo ra khi dch nui cy bng cch ly tm v lm sch t bo. Thng thng khong 1.000ml dch nui cy li tm v thu ly khong 10ml cn vi khun. 2. Ph v mng t bo v gii phng cc thnh phn bn trong Axt nucleic c gii phng ra khi mng t bo bng phng php ha hc v sinh hc thu dch chit t bo. Enzyme lysozyme l mt enzyme c trong lng trng trng hoc phage T4, c kh nng ph v thnh phn trng hp ca thnh t bo. EDTA (TrilonB) to phc vi Mg+2 dng ha tan v lm v thnh t bo. ng thi EDTA ngn khng cho enzyme trong t bo phn hy DNA. Ngi ta cn c th ph v mng t bo v gii phng DNA ra khi lin kt histon bng cht ty ra SDS (Sodium Derecyl Sylfat) hoc enzyme protease. Cc cht ph v mng ny lm li gii mng, tch ri cc phn t lipit v gy ra s gy mng t bo. 3. Lm sch DNA t dch chit t bo Loi b cc thnh phn khng mong mun nh protein, lipit v cc thnh phn khc bng kt ta phn on. Mu c lc mnh trong h dung mi phenol-chloroform bin tnh, kt ta protein v gii phng axt nucleic vo dung dch lng. Protein b bin tnh s tch ra khi pha nc c cha DNA s c tch ra nh ly tm. Dch ni gm axit nucleic v nc c ht ra v tip tc phn hy RNA bng enzyme RNase. Sau , tch DNA v nc thc hin bc phn tch tip theo. 4. Thu hi DNA dng c Mc ch ca bc ny l thu DNA dng c c, nhm bo v chng khi s phn hy ca enzyme. C 2 phng php kt ta:

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7/24/2012 1:37 AM

Phng php tch chit DNA v plasmid ca vi khun

file:///C:/Users/socnhim/Desktop/5219-phuong-phap-tach-chiet-dna-va-p...

+ - Kt ta DNA trong etanol vi s c mt Na nng cao, nhit thp, t l gia etanol v mu phn tch l 3:1. - Kt ta trong izopropanol, t l gia ru v mu l 1:1. Vi phng php ny khng cn s c mt ca mui v loi c DNA c phn t lng thp. Ly tm cao tc v thu nhn DNA dng c c. Trong c hai phng php, DNA thu c s ra trong etanol-70% loi izopropanol v mui thu c DNA tinh khit. II. Phng php tch DNA plasmid Vic tch DNA plasmid ra khi DNA nhim sc th l rt kh khn. Tuy nhin ngi ta c th da vo s khc nhau l ha gia hai loi DNA nh: kch thc, hnh thi, cu trc ca chng. V nguyn tc c tin hnh theo 3 bc sau: CV1 - Cao ng Ni v H Ni 2011 BI MI NHT CHUYN BI GING - Bc 1 : Sau khi thu hi v lm sch cc t bo c cha plasmid, ngi ta x l trong hn hp CHH - Cao ng Hng Hi 2011 SDS, EDTA hoc NaOH lm DNA si i bin tnh thnh DNA si n nm cnh nhau. QHS - i Hc QG H Ni - i hc Gio dc 2011 - Bc 2: X l hn hp trn trong mi trng m (NaCH3COO+CH3COOH) kt ta protein v THP - i hc Hi Phng 2011 SDS dng tp hp. YPB - i hc Y Hi Phng 2011 - Bc 3: a v mi trng trung tnh, DNA si n c hi tnh tr li. Cn DNA (NST) do diTDL - i hc Lt 2011 nn hi tnh chm v kt ta cng SDS. Tch DNA plasmid bng cch kt ta trong etanol hoc CPN - Cao ng Phng ng - Nng 2011 izopropanol, sau ly tm tch c plasmid tinh sch. CKY - C K thut Y T II 2011 Theo CNSH YTC - i hc Y t Cng Cng 2011 CDD - Cao ng Dn Lp Kinh T K Thut ng Du Nng 2011

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7/24/2012 1:37 AM

Phng php tch chit DNA v plasmid ca vi khun

file:///C:/Users/socnhim/Desktop/5219-phuong-phap-tach-chiet-dna-va-p...

Phng php tch RNA tng s v mRNA Cc bc c bn ca phng php tch dng Cc phng php to ADN ti t hp Cc cng on chnh to ADN ti t hp ADN ti t hp Vector chuyn gen l NST nhn to ca nm men v ng vt c v Vector chuyn gen l cosmid Cc vector chuyn gen l phage Tin mi hn: Th nghim bt ng t bo thc vt Th nghim ng cong sinh trng ca t bo thc vt Th nghim ng cong sinh trng ca nm men Bt ng t bo Xc nh sinh trng ca t bo Cc phng php lai phn t u, nhc im v ng dng ca phng php PCR Cc yu t nh hng n phn ng PCR Phng php PRC v cc nguyn tc thc hin Tch v thu nhn gen Tin c hn: Ngn hng cDNA Ngn hng gen (Genomic library) Mt s phng php xc nh dng cn tm Mt s phng php a ADN ti t hp vo t bo ch Vector chuyn gen l plasmid Vector chuyn gen Cc loi enzim tng hp axit nuclic Enzyme gii hn v h thng hn ch - ci bin (Restriction modification system) Tnh c hiu v tr ca enzim ct gii hn - RE - Rectriction Enzyme Enzim gii hn << Trang truc Trang k >>

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