Gene Cloning 분자생물학실험 이효민

DNA Cloning DNA cloning is a technique for reproducing DNA fragments. It can be achieved by two different approaches: a.cell based b.using polymerase chain reaction (PCR). a vector is required to carry the DNA fragment of interest into the host cell.

1.total RNA preparation from C. elegans 2.cDNA synthesis by RT 3.PCR 4.Ligation of DNA fragment with T vector 5.transformation in DH5α competent cell 6.Isolation of plasmid and enzyme digestion Gene Cloning 의 과정

RT-PCR (Reverse Transcriptase Polymerase chain reaction) mRNAcDNAPCR Reverse transcriptase Taq polymerase

PCR 1. Taq polymerase Thermus aquaticus 에서 분리한 polymerase Thermo-stable 한 enzyme 5’ →3’ polymerization activity 를 가짐 (3’→5’ exonuclease activity 를 가지지 않음 ) 60 bp/sec 의 합성능력을 가짐 PCR product 의 3’end 에 A overhang 을 만듦 → TA cloning 에 이용되는 성질

TA cloning

PCR 2. dNTP mix dNTP ( dATP, dTTP, dGTP, dCTP) PCR 시에 필요한 nucleotide 공급 3. 10X buffer Magnesium ion (Mg 2+ ) KCl Gelatin 또는 bovine serum albumin(BSA)

PCR STEP Denaturation : denaturation of DNA due to high temperatures is the separation of a double strand into two single strands / 94 ℃ Annealing : the primers to bind to their complementary DNA sequences / Tm = 4*(G+C)+2*(A+T) annealing 온도는 Tm 값보다 2~5 ℃ 낮게 한다 Elongation : taq-polymerase synthesis polynucleotide