User:Vincent Chiang/Notebook/TA Cloning for sequencing/2014/10/27

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TA Cloning

Objective: In order to check the sequences of Class C β-lactamase (ANICBIBUN_05775, ANICBIBUN_05775) and vaccine candidates (AdeI, A1S_2023), we conducted TA cloning for further sequencing.

Materials: 1. TOPO TA cloning kit (Invitrogen, 25-0185I) 2. Phusion, High-Fidelity DNA polymerase (F-530L, FINNZYMES)[[1]] 3. A. nosocomilis strains: 345, 655, 1558 4. A. baumannii strains: ATCC 17978

Protocol: 1. An & Ab strains preparation: To scratch colony to 300 μL ddH2O, boiling to 100 °C for 10 min, centrifuge to collect supernatant. To dilute the con. to about 100 ng/ml. 2. Using proofreading Taq polymerase for sequence fidelity. 3. PCR Component: PCR condition: Templates 2μL 98°C 30" 5X PCR buffer 10μL 98°C 10" 10μM dNTPs 1μL 53°C 30" Primer F 0.5μL (50°C for A1S_2023)

      R       0.5μL              72°C           40"

DMSO 1.5μL 72°C 10' Taq pol. 0.5μL 30 cycles ddH2O 34μL Total Vol. 50μL