제한 효소pta322 ecori 처리 방법

Cloning�� ܰ�, ó��̶�� ƽ��ϴ�.
�ܰ� �ܰ� �� ư��ٸ�, ��ŵ� Cloning �� ֽ��ϴ�.

Cloning �̶�?

Plasmid (vector) �� Ű�ü�� ̿��Ͽ� ��ϴ� �� (insert) �� Ű�� ڻ�� , ��ڸ� �� vector�� ϰ� ��ϴ� �Ϸ�� cloning�̶�� Ī�մϴ�. Cloning �� ڰ��н�� ϳ�� ȿ�Ұ� �߰ߵǰ� ��ڻ�� 迡 ��ʿ� �� 뼺�� θ� �̿�Ǿ� ��, �ٷ�� ȿ�� ó�� ϴ� cloning �� (TA cloning, PCR cloning ��)�� Ȱ�� ֽ��ϴ�.
Cloning�� ̿�� insert DNA�� ÷κ�� genomic DNA ��⡤��ϰų�, RNA�� cDNA�� ռ�, �Ǵ� PCR�� DNA �� ϴ� �� Ȯ�� ֽ��ϴ�. ��Ȥ insert DNA�� vector�� ȿ�� ʴ� ��, PCR primer�� ȿ�� Ʈ�� ߰��Ͽ� �� , insert DNA�� PCR ��Ͽ� cloning�� ʿ�� ȿ�� Ʈ�� insert DNA�� ̴� �� ϱ⵵ �մϴ�.

�� Cloning ��̵忡�� θ� ��ϴ� �� ߿� �ϳ��

(1) TA cloning

��

(2) ��ȿ�� ligation�� ̿�� cloning

�� TA cloning�� Ȯ�� clone�� ȿ�� ó�� insert�� и��Ͽ� �� vector�� ű�� ÷� �� Ұ��ϰ�� մϴ�.

[Cloning Workflow]

TA-cloning�� ȿ�Ҹ� �̿�� cloning

�Ϲ�� Cloning�� insert DNA�� vector�� ȿ�ҷ� �ڸ�� , ligase �� ִ� �� մϴ�. �� vector�� MCS �� ȿ�� (insert DNA�� ʴ� ��ȿ��)�� ϸ�, �� ȿ�� ڸ�� insert DNA�� PCR�� primer�� ȿ�� ߰��Ͽ� ��ν� insert DNA �� ܿ� ��ȿ�� Ʈ�� ֽ��ϴ�.
�� PCR�� ΰ�� ȿ�� Ʈ�� DNA�� ܿ� ��ϹǷ� ��ȿ�� ó�� ȿ�� ְ�, insert DNA�� ȿ�� ó��/agarose gel �� ġ�� ʱ⿡ �� DNA�� Ȯ��ؾ� �ϴ� �� ߻�� ֽ��ϴ�. �� PCR �깰�� Ȯ��ϱ� �� PCR �� Ŭ�� ø��⵵ ��, �� insert DNA �� PCR �� ߻�� Ȯ�� Բ� ��Ƿ�, ��Ȯ�� insert DNA�� Ȯ��ؾ� �ϴ� cloning ��迡 ��õ�� ʽ��ϴ�.
�� PCR�� insert DNA�� ⼭�� Ȯ�� (sequencing)�ϰ�, ��Ȯ�� insert DNA�� ټ� Ȯ�� ֵ�� PCR �깰�� ٷ� TA-cloning �ϴ� �� ̿�� ֽ��ϴ�.
TA-cloning�� insert DNA�� Ե� plasmid�� Ȯ��ϸ�, circular �� plasmid�� Ƿ�, ��ȿ�� ܽ� �� ȿ�� , ��Ȯ�� ȿ�� Ʈ�� insert DNA�� Ȯ�� ־� �� vector�� cloning ȿ�� ִ� �� ֽ��ϴ�.
�Ʒ�� High Fidelity PCR ȿ�Ҹ� �̿��Ͽ� insert DNA�� TA-cloning�� vector�� sub-cloning�ϴ� �� Ұ��մϴ�.

    - TA-cloning�� ȿ�Ҹ� �̿�� cloning (Ŭ��Ͽ� �� Ȯ��ϼ��)
       1. Insert DNA (��) PCR
           - PCR�� Ȯ�� PCR�깰 ��ܿ� ��Ͽ�
       2. PCR �깰 ��
       3. PCR ��깰�� TA cloning
       4. E. coli ��ȯ (transformation)
       5. Insert Ȯ��

�� PCR ȿ�� Fidelity (��Ȯ��) �� PCR �깰 ��ܿ� ��Ͽ�

��ȿ�� ó�� vector�� sub-cloning

�Ϲ�� TA-cloning�� Ȯ�� recombinant clone�� sequencing �Ŀ�, �� ϴ� vector�� (insert)�� ű�� ϰ� �˴ϴ�.
��, ��ڸ� ��Ű�� ϴ� �� پ�� vector�� ֽ��ϴ�.

�پ�� viral delivery system : Lentivirus, Adeno-associated virus(AAV), Retrovirus, Adenovirus - �ٷΰ��
CRISPR/Cas9 genome editing system : Plasmdi, virus, single-cell cloning �ý�� - �ٷΰ��
�Ĺ�� binary vector : Agrobacterium �Ű� �Ĺ��ȯ�� vector �� - �ٷΰ��
E. coli protein expression vector : cold shock vector, Chaperone �� C.cell �� - �ٷΰ��

��⼭�� 1) Adeno-associated virus system (AAV2)�� AAV vector�� sub-cloning �� 2) �Ĺ��ȯ�� binary vector�� sub-cloning�� ÷� �Ұ��մϴ�.

   - ��ȿ�� ó�� vector�� sub-cloning (Ŭ��Ͽ� �� Ȯ��ϼ��)
       1. Expression vector �Ǵ� �� vector �� insert DNA �غ�
       2. Ligation �� E. coli ��ȯ (transformation)
       3. Insert Ȯ��

�� ȿ�� ó��, �� ʾƿ�!

�� ñ��ؿ�~ Ż�λ�ȭȿ�� (Alkaline Phosphatase)

Cloning - FAQs

Q1. Ligation�̳� ��ȯ ȿ�� ִ� �� Դϱ�?
A1. �� 並 ��õ�մϴ�.

Ligation �� ð�� ÷��ݴϴ�.
DNA ��׳�� (salt) �󵵰� �� ligation ȿ�� ϵǸ�, Ư�� ź�� ħ�� Ǵ� ammonium acetate�� ligation �� ۿ�� ϱ� ��, ��ź�� ħ�� ʰ� �� washing �ϴ� �� ߿��մϴ�.
DNA �� kit�� ϴ� ��, �� ź�÷� ħ��ϰ� ��۸� ��ȯ�ϸ� ligation ȿ�� ֽ��ϴ�.
��⸻�� (cohensive end) ligation�� DNA �� (vector+insert DNA)�� 60~65�ɿ�� 2~3 �� incubation ��, �ٷ� �� ¿�� ligation �þ�� ÷��Ͽ� �� ϸ�, ligation�� ȿ�� ⸻�� Ȯ��Ǿ� ligation ȿ�� ֽ��ϴ�.

�� ligation ȿ�� 쿡�� DNA �� ٽ� ��ϴ� �� մϴ�.

Q2. �� cloning�� Դϱ�?
A2. ��̰� �� ligation�� ȿ�� ϵǴ� �� ֽ��ϴ�. TaKaRa DNA Ligation Kit LONG (Code 6024)�� ligation�� ȭ�Ǿ� �־�, 10 kb �̻�� ligation�� ϴ� ��쿡 ��մϴ�. �� Insert DNA�� plasmid�� ü ��̰� ū �� (Ư��, 10 kb�� Ѵ� ��)�� ȿ�� , �� plasmid �� Ƿ�, ��Ȯ�� clone�� Ȯ��ϱ� ��ϴ�. �̷�� ū �� DNA�� ȿ�� ȯ�� ִ� E. coli HST08 Premium Competent Cells(Code 9128)�� մϴ�.

Cloning �� ǰ ��Ʈ

��Ȯ�� ȿ�� , Cloning�� PCR ȿ�� - PrimeSTAR�� GXL DNA Polymerase
Colony PCR�� premix - EmeraldAmp�� GT PCR Master Mix
��ȿ�� Ligation premix - DNA Ligation Kit ��Mighty Mix��
Mighty TA-cloning Kit - Mighty TA-cloning Kit
Blunt end PCR �깰�� TA cloning�� - Mighty TA-cloning Reagent Set for PrimeSTAR��
�پ�� Ż�λ�ȭȿ�� - Alkaline Phosphatase (Calf intestine) / Alkaline Phosphatase (E. coli C75)
��ȿ�� Competent Cell - E. coli Competent Cells/ E. coli Electro-Cells
�� down!, �� up!! ��׷��̵� PCR cloning kit - In-Fusion�� Snap Assembly Master Mix