"

From 2010.igem.org

PCR with Pho DNA Polymerase (NIPPON GENE) ＜Materials＞ 10x reaction buffer Forward primer (starting with 20µM) Reverse primer (starting with 20µM) Pho DNA Polymerase (starting with 2.5U/µl) dNTP (starting with 2.5mM) DW

 DNA samples
   ・Pellet of E-coli having BBa_I723901

　　・Pellet of E-coli having BBa_K208017 　・Plasmid of BBa_I13521(starting with 20nM)

   ・Pellet of JM109

<Protocol> 1 Make the pellet with pre-colony. 2 Thaw all required reagents completely and put them on ice. Mix all reagents well by inversion and spin them down prior to pipeting. 3 Prepare the reaction mix to correct for dispensing losses prepare an excess of reaction mix (for example, a 100 reactions mix for 96 reactions) 10x reaction buffer 12.5μl Forward primer 2.5μl (starting with 20µM) Reverse primer 2.5μl (starting with 20µM) Pho DNA Polymerase 2.5µl (starting with 2.5U/µl) dNTP 20μl (starting with 2.5mM) DW 85µl Total reaction Mix 125μl 4 Add all components together, except for the template. Mix thoroughly by inversion. Spin down. 5 Add the template DNA(pellet or Plasmid) for your samples in to PCR tubes 6 Add 50μl of the reaction mix per tube and mix gently on a stirrer or spin down. Ensure that no bubbles are present in the reaction tube. Reaction set up can be done at room temperature. 7 Program your Real-Time Thermocycler w/ “fast block” using the following recommended FAST parameters: 95°C 5min.

95°C       30sec.              30cycle
55°C       30sec.              30cycle
76.5~68.0°C 3min and 45sec.    30cycle

72°C 5min.

     4°C    

Ligation with Ligation-convenience kit (NIPPN GENE) <Materials>

 DNA solution
 2 × Ligation Mix (Ligation-convenience kit from NIPPN GENE)

<Protocol> 1 Prepare 10 μl of DNA solution to contain DNA fragment with appropriate mole ratio against vector DNA. 2 Add 10μl of 2 × Ligation Mix to the DNA solution and mix well. 3 Ligation reaction. Incubate 5-30min at 16°C. 4 Apply DNA reaction mixture directly to transformation or in vitro packaging as it is.

DNA extraction with ISOPLANT(NIPPON GENE) ＜Materials＞

Extraction Buffer 0.3ml
Lysis Buffer 0.15ml
Sodium Acetate(pH5.2) 0.15ml
10mM Tris-HCl(pH8.0), 1mM EDTA 0.1ml
1mg/ml RNaseA 1µl
E-coli culture
H20
TE Buffer

＜Protocol＞ 1 Centrifuge 1.5ml of E-coli culture at 4οC for 5min, and then aspirate the supernatant. 2 Add 0.3ml of Extraction Buffer to sample, and then vortex 1~2sec. 3 Add 0.15mlof Lysis Buffer to solution, and then vortex 5~6sec. 4 Incubate 15min at 50°C. 5 Add 0.15ml of Sodium Acetate(pH5.2) to solution, and then vortex 1~2sec. 6 Leave on ice for 15min 7 Centrifuge solution at 4οC for 15min, and then aspirate the supernatant. 8 Add 0.3ml of H2O to the tube having the pellets, and then vortex thoroughly. 9 Add 0.6ml of EtOH to solution, and then vortex thoroughly. 10 Centrifuge solution for 10min, and then aspirate the supernatant. 11 Add 70%EtOH to the tube having the pellets. 12 Dry the pellets in the speed-vac for 5min, or air dry. 13 Add TE Buffer to the dried pellets