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	<h3>LB medium (1 L)</h3>		<h3>LB medium (1 L)</h3>
-	1; Add about 900 mL of distilled water to beaker.	+	1; Add about 900 mL of distilled water to beaker.<br>
-	2; Add reagents (Table) and stir.	+	2; Add reagents (Table) and stir. <br>
-	3; Add distilled water up to 1 L and take LB medium to media bottle.	+	3; Add distilled water up to 1 L and take LB medium to media bottle.<br>
-	4; Autoclave for 20 min at 120°C	+	4; Autoclave for 20 min at 120°C.<br>
	<h3>LB agar gel (1 L)</h3>		<h3>LB agar gel (1 L)</h3>
-	1; Prepare LB medium without autoclave (Steps 1-3 of 1L scale of LB medium).	+	1; Prepare LB medium without autoclave (Steps 1-3 of 1L scale of LB medium).<br>
-	2; Add 15 g of agar and stirrer bar.	+	2; Add 15 g of agar and stirrer bar.<br>
-	3; Autoclave for 20 minutes at 120°C.	+	3; Autoclave for 20 minutes at 120°C.<br>
-	4; Stir and cool LB medium with agar, add appropriate antibiotic (50 μL).	+	4; Stir and cool LB medium with agar, add appropriate antibiotic (50 μL).<br>
-	5; Pour LB medium (Step 4) in plate and cool down in clean bench.	+	5; Pour LB medium (Step 4) in plate and cool down in clean bench.<br>
	<h2>Transformation</h2>		<h2>Transformation</h2>
	<h3>Preparation of E. coli contain particular plasmid</h3>		<h3>Preparation of E. coli contain particular plasmid</h3>
-	1; Incubate frozen competent cell (DH5α) on the ice for a few minutes.	+	1; Incubate frozen competent cell (DH5α) on the ice for a few minutes. <br>
-	2; Add 1~5 μL of plasmid to competent cell (DH5α) on the ice.	+	2; Add 1~5 μL of plasmid to competent cell (DH5α) on the ice.<br>
-	3; Incubate for 20 – 30 minutes on the ice.	+	3; Incubate for 20 – 30 minutes on the ice.<br>
-	4; Incubate for 45 seconds at 42°C.	+	4; Incubate for 45 seconds at 42°C.<br>
-	5; Add 1 mL LB medium and cultivate for 1 hour at 37°C.	+	5; Add 1 mL LB medium and cultivate for 1 hour at 37°C.<br>
-	6; Spread culture medium on LB agar plate with appropriate antibiotic.	+	6; Spread culture medium on LB agar plate with appropriate antibiotic.<br>
	<h2>Plasmid extraction</h2>		<h2>Plasmid extraction</h2>
	<h3>Preparation of plasmid extracted from <i>E. coli</i></h3>		<h3>Preparation of plasmid extracted from <i>E. coli</i></h3>
-	1; Pick up single colony from agar plate and cultivate it in 1.5 mL LB medium containing appropriate antibiotec (50 μg/mL) overnight at 37°C.	+	1; Pick up single colony from agar plate and cultivate it in 1.5 mL LB medium containing appropriate antibiotec (50 μg/mL) overnight at 37°C.<br>
-	2; Move the culture medium to 1.5 mL tube.	+	2; Move the culture medium to 1.5 mL tube.<br>
-	3; Centrifuge for 5 seconds at 15,000×g and 4 °C and discard supernatant.	+	3; Centrifuge for 5 seconds at 15,000×g and 4 °C and discard supernatant.<br>
-	4; Add 100 μL Solution 1 to the pellet and resuspend and incubate for 3 minutes.	+	4; Add 100 μL Solution 1 to the pellet and resuspend and incubate for 3 minutes.<br>
-	5; Add 100 μL Solution 2, invert tube gently 5 times and incubate for 3 minutes.	+	5; Add 100 μL Solution 2, invert tube gently 5 times and incubate for 3 minutes.<br>
-	6; Add 100 μL Solution 3, invert tube 5 times and incubate for 3 minutes.	+	6; Add 100 μL Solution 3, invert tube 5 times and incubate for 3 minutes. <br>
-	8; Add 200 μL Solution 4 and invert tube 5 times and centrifuge for 3 minutes at 15,000×g and 4 °C.	+	8; Add 200 μL Solution 4 and invert tube 5 times and centrifuge for 3 minutes at 15,000×g and 4 °C.<br>
-	10; Take supernatant to new 1.5 mL tube and centrifuge for 3 minutes at 15,000×g and 4 °C.	+	10; Take supernatant to new 1.5 mL tube and centrifuge for 3 minutes at 15,000×g and 4 °C.<br>
-	11; Vortex Bind mix for 1 min and add 800 μL Bind mix to new 1.5 mL tube.	+	11; Vortex Bind mix for 1 min and add 800 μL Bind mix to new 1.5 mL tube.<br>
-	12; Add 400 μL supernatant after centrifugation (Step 10) to tube containing Bind mix (Step 11) and mix.	+	12; Add 400 μL supernatant after centrifugation (Step 10) to tube containing Bind mix (Step 11) and mix.<br>
-	13; Incubate for 3 minutes, centrifuge for 3 seconds at 5,000×g and 4 °C, and discard supernatant.	+	13; Incubate for 3 minutes, centrifuge for 3 seconds at 5,000×g and 4 °C, and discard supernatant.<br>
-	14; Add 1 mL of 50% ethanol and resuspend.	+	14; Add 1 mL of 50% ethanol and resuspend. <br>
-	15; Centrifuge for 3 seconds at 5,000×g and 4 °C and discard supernatant.	+	15; Centrifuge for 3 seconds at 5,000×g and 4 °C and discard supernatant.<br>
-	16; Repeat wash (Steps 14-15).	+	16; Repeat wash (Steps 14-15).<br>
-	17; Dry pellet for a few minuet under a vacuum to remove residual ethanol.	+	17; Dry pellet for a few minuet under a vacuum to remove residual ethanol.<br>
-	18; Add 50 μL nuclease-free water or TE buffer and incubate for 3 minutes at 65°C.	+	18; Add 50 μL nuclease-free water or TE buffer and incubate for 3 minutes at 65°C.<br>
-	19; Centrifuge for 3 minutes at 15,000×g and 4 °C.	+	19; Centrifuge for 3 minutes at 15,000×g and 4 °C.<br>
-	19; 40 μL of supernatant into new 500 μL tube .	+	19; 40 μL of supernatant into new 500 μL tube.<br>
	<h2>Restriction enzyme digestion of DNA</h2>		<h2>Restriction enzyme digestion of DNA</h2>

---

Revision as of 15:05, 27 October 2010

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Protocols

LB medium and LB agar gel Medium for cultivation of E. coli

LB medium (1 L)

1; Add about 900 mL of distilled water to beaker.
2; Add reagents (Table) and stir.
3; Add distilled water up to 1 L and take LB medium to media bottle.
4; Autoclave for 20 min at 120°C.

LB agar gel (1 L)

1; Prepare LB medium without autoclave (Steps 1-3 of 1L scale of LB medium).
2; Add 15 g of agar and stirrer bar.
3; Autoclave for 20 minutes at 120°C.
4; Stir and cool LB medium with agar, add appropriate antibiotic (50 μL).
5; Pour LB medium (Step 4) in plate and cool down in clean bench.

Transformation

Preparation of E. coli contain particular plasmid

1; Incubate frozen competent cell (DH5α) on the ice for a few minutes.
2; Add 1~5 μL of plasmid to competent cell (DH5α) on the ice.
3; Incubate for 20 – 30 minutes on the ice.
4; Incubate for 45 seconds at 42°C.
5; Add 1 mL LB medium and cultivate for 1 hour at 37°C.
6; Spread culture medium on LB agar plate with appropriate antibiotic.

Plasmid extraction

Preparation of plasmid extracted from E. coli

1; Pick up single colony from agar plate and cultivate it in 1.5 mL LB medium containing appropriate antibiotec (50 μg/mL) overnight at 37°C.
2; Move the culture medium to 1.5 mL tube.
3; Centrifuge for 5 seconds at 15,000×g and 4 °C and discard supernatant.
4; Add 100 μL Solution 1 to the pellet and resuspend and incubate for 3 minutes.
5; Add 100 μL Solution 2, invert tube gently 5 times and incubate for 3 minutes.
6; Add 100 μL Solution 3, invert tube 5 times and incubate for 3 minutes.
8; Add 200 μL Solution 4 and invert tube 5 times and centrifuge for 3 minutes at 15,000×g and 4 °C.
10; Take supernatant to new 1.5 mL tube and centrifuge for 3 minutes at 15,000×g and 4 °C.
11; Vortex Bind mix for 1 min and add 800 μL Bind mix to new 1.5 mL tube.
12; Add 400 μL supernatant after centrifugation (Step 10) to tube containing Bind mix (Step 11) and mix.
13; Incubate for 3 minutes, centrifuge for 3 seconds at 5,000×g and 4 °C, and discard supernatant.
14; Add 1 mL of 50% ethanol and resuspend.
15; Centrifuge for 3 seconds at 5,000×g and 4 °C and discard supernatant.
16; Repeat wash (Steps 14-15).
17; Dry pellet for a few minuet under a vacuum to remove residual ethanol.
18; Add 50 μL nuclease-free water or TE buffer and incubate for 3 minutes at 65°C.
19; Centrifuge for 3 minutes at 15,000×g and 4 °C.
19; 40 μL of supernatant into new 500 μL tube.

Restriction enzyme digestion of DNA

Cleavage of insert DNA from plasmid

1; Mix DNA and restriction enzyme (Table).
2; Incubate for 2 hours at 37°C.
3; Incubate for 10 minutes at 65°C.
4; Confirm the band of DNA by agar gel electrophoresis.

Confirmation and separation of digested DNA

Preparation of agar gel

1; Add 1 g of agar to 100 mL of 1×TAE.
2; Boil and stir until solution is clear.
3; Cool down, pour to gel form and set gel corm.
4; Incubate until gel dry out.
5; Stare gel in 1×TAE.

Agar gel electrophoresis

1; Place agar gel and pour 1×TAE in electrophoresis chamber.
2; Load DNA ladder and DNA sample mixed with loading dye on agar gel.
3; Electrophorese for 20 minutes at 100 V.
4; Stain gel by Sybr SafeTM Gel Stain (Invitrogen).
5; Visualize the band of DNA using UV light.
6; Confirm the length of digested DNA.

Gel purification

Purification of DNA from agar gel

GENECLEAN® II Kit(NaI、glass milk、NEW Wash)/Qbiogene

1; Cut the objective band in the agar gel after electrophoresis and stain with SYBR Safe.
2; Put the gel including objective DNA into 1.5 mL tube and measure the mass of that.
3; Add 2.5-3 fold volume NaI solution into the tube (Gel: NaI =1 mg : 1 μL ).
4; Incubate the gel at 50°C for 5 minute.
5; Add 10 μL of glass milk and vortex.
6; Incubate for 5 minutes and vortex per a minute.
7; Centrifuge for 5 seconds at 15,000×g and 4°C and discard the supernatant.
8; Add the 500μL of New Wash and resuspend.
9; Centrifuge for 5 seconds at 15,000×g and 4°C.
10; Repeat wash (Steps 8-9).
11; Dry the pellet for 5-10 minutes under vacuum.
12. Add 20　μL of nuclease-free water and resuspend.
13. Centrifuge for 5 seconds at 15,000×g and 25°C.
14. Transfer supernatant including objective DNA into new tube.

Ligation

Ligation inset DNA and vector
DNA Ligation kit Ver 2.1(SolutionⅠ)/Takara

1; Mix the insert DNA, vector and solution I (Table).
2; Incubation at 16°C for 30 minute.
3; Transform E. coli with ligation sample.

Colony PCR

Confirm of insert DNA in plasmid, directly using E. coli at PCR.
1; Add 10 μL of reagent solution (Table 1) to PCR tube.
2; Pick up single colony from agar plate with tooth pick and sting replica plate (new LB agar plate).
3; Put and stir toothpick to reagent solution (Step 1).
4; Amplify insert DNA with PCR program (Table 2).
5; Electrophorese PCR sample with agar gel.
6; Check the band and length of insert DNA and decide the colony with insert DNA.

Sequence analysis

Identification of insert DNA
*Preparation of PCR product
Big Dye® Terminator Cycle Sequencing Kit Ver. 3.1 (Premix, Buffer) / Applied Biosystems
1; Add reagent solution (Table 1) to PCR tube and amplify insert DNA with PCR program
(Table2). *Purification of PCR product and sequence analysis
Agencourt CleanSEQ® and 96 R ring Super Magnetic Plate® / Beckman Coulter
1; Add 10 μL of Agencourt CleanSEQ® 10 µL to PCR product.
2; Add 62 μL of 85% ethanol, mix and incubate for 3 minutes.
3; Incubate for 3 minutes on 96 R ring Super Magnetic Plate® and discard supernatant.
4; Add 100 μL of 85% ethanol and mix.
5; Incubate for 3 minutes on 96 R ring Super Magnetic Plate® and discard supernatant.
6; Repeat wash (Steps 4-5).
7; Dry for 10 minutes.
8; Add 40 μL nuclease-free water and mix.
9; Transfer 30μL of clear sample into a new plate for loading on the detector.
10; Load sample on sequencer and analyze.

Time schedule

• June
• July
• August
• September
• October
• November

Progress in this year

Future plan

Retrieved from "http://2010.igem.org/Team:Tokyo-NoKoGen/experiments"