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From 2010.igem.org

PCR with Pho DNA Polymerase (NIPPON GENE)

<Materials>

10x reaction buffer</br>
Forward primer (starting with 20µM)</br>
Reverse primer (starting with 20µM)</br>
Pho DNA Polymerase (starting with 2.5U/µl)</br>
dNTP (starting with 2.5mM)</br>
DW</br>
DNA samples</br>
   ・Pellet of E-coli having BBa_I723901</br>

　　・Pellet of E-coli having BBa_K208017</br>

 　・Plasmid of BBa_I13521(starting with 20nM)</br>
   ・Pellet of JM109</br>

<Protocol></br>

1 Make the pellet with pre-colony.</br>
2 Thaw all required reagents completely and put them on ice. Mix all reagents well by inversion and spin them down prior to pipeting.</br>
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)</br>
    10x reaction buffer 12.5μl</br>
    Forward primer 2.5μl (starting with 20µM)</br>
    Reverse primer 2.5μl (starting with 20µM)</br>
    Pho DNA Polymerase 2.5µl (starting with 2.5U/µl)</br>
    dNTP 20μl (starting with 2.5mM)</br>
    DW 85µl</br>
    Total reaction Mix 125μl </br>
4 Add all components together, except for the template. Mix thoroughly by inversion. Spin down.</br>
5 Add the template DNA(pellet or Plasmid) for your samples in to PCR tubes</br>
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.</br>
7 Program your Real-Time Thermocycler w/ “fast block” using the following recommended FAST parameters:</br>
    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 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