1. Gene Cloning 분자생물학실험 이효민 2012-9-7

2. 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.

3. 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

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

5. 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

6. TA cloning

7. 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)

8. 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