Team:HokkaidoU Japan/Notebook/ecoli

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• E. coli
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E. coli

March

2015/03/10

Competent Cells

mami

BL21 (DE3) pLysS

1. Thawed original competent cells (BL21 (DE3) pLysS) on ice.
2. Added 5 μL of original competent cells to 2 mL of LB.
3. Incubated the cells for 16 hrs at 37℃.
4. Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.
5. Incubated the cells at 130 rpm for 14 hrs at 20℃, until OD₆₀₀ reach 0.5.
6. Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
7. Removed supernatant and added 75 mL of TB to each tube.
8. Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
9. Removed supernatant and added 32 mL of TB.
10. Added 32 μL of DMSO 10 times.
11. Took 50 μL and froze with liquid nitrogen.

2015/03/11

PCR

実験者

BBa_R0011

Reagent	Volume
BBa_R0011	1 μL
100bpUP-EX-F 10 μM	1.5 μL
200bpDN-PS-R 10 μM	1.5 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	32 μL
Total	50 μL

BBa_0030 - BBa_E1010

Reagent	Volume
BBa_0030 - BBa_E1010	1 μL
100bpUP-EX-F 10 μM	1.5 μL
200bpDN-PS-R 10 μM	1.5 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	32 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	62.6℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	1 min	Elongation	30 cycle
Store	4℃	Hold	Store

PCR Purification

実験者

BBa_R0011, BBa_0030 - BBa_E1010
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Digestion

実験者

BBa_R0011

Reagent	Volume
BBa_R0011	44 μL
XbaI	1 μL
Cut Smart	5 μL
Total	50 μL

BBa_0030 - BBa_E1010

Reagent	Volume
BBa_0030 - BBa_E1010	44 μL
XbaI	1 μL
Cut Smart	5 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	300 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

実験者

BBa_R0011, BBa_0030 - BBa_E1010

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	2x TBE

Gel Extract

実験者

BBa_R0011, BBa_0030 - BBa_E1010
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Electrophoresis

実験者

BBa_R0011, BBa_0030 - BBa_E1010

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	2x TBE

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