Team:Tokyo-NoKoGen/protocols

From 2011.igem.org

'''Protocols'''

Tokyo-NokoGen 2011

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology

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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 25 g of LB medium, Miller(MERCK) 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 (table).
5; Pour LB medium (Step 4) in plate and cool down in clean bench.

	f.c.
Ampicillin	100 μg/mL
Kanamycin	50 μg/mL
Chloramphenicol	30 μg/mL
Tetlacycline	11 μg/mL

Transformation

Inserting plasmid into E. coli

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 antibiotic 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.

reagent name	volume
DNA restriction enzyme A restriction enzyme B buffre MQ	5 μL 0.5 μL 0.5 μL 1.5 μL 7.5 μL
total	15 μL

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 dissolved and clear.
3; Cool down, pour into container to set its shape.
4; Wait until gel dries.
5; Store 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.

PCR

1; Add 25 μL of reagent solution (Table 1) to PCR tube.
2; Amplify target DNA with PCR program (Table 2).
3; Confirm the band of DNA by agar gel electrophoresis.

reagent name	volume
primeSTAR® GXL DNA polymerase 5X GXL buffer dNTP template DNA fowerd primer reverse primer MQ	0.5 μL 5 μL 2 μL 1 μL 1 μL 1 μL 14.5 μL
total	25 μL

Table 1

	Step 1	Step 2	Step 3
Cycle	1	30	1
	98℃ 1:00	98℃ 0:10 68℃ 55℃ 4:00 0:10	68℃ 2：00 4℃ ∞

Table2

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.

reagent name	volume
insert DNA vector solution I	2 μL 2 μL 4 μL
total	8 μL

Colony PCR

Confirmation of insert DNA in plasmid, directly doing PCR on E. coli
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.

reagent name	volume
fowerd primer reverse primer Go taq® Green Master Mix(Promega) MQ	0.5 μL 0.5 μL 5 μL 4 μL
total	10 μL

Table 1

	Step 1	Step 2	Step 3
Cycle	1	30	1
	95℃ 1:00	95℃ 0:10 72℃ 55℃ 4:00 0:10	72℃ 2：00 4℃ ∞

Table2

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.

reagent name	volume
plasmid primer premix buffer MQ	3 μL 0.5 μL 0.5 μL 4 μL 12 μL
total	20 μL

Table 1

	Step 1	Step 2	Step 3
Cycle	1	30	1
	95℃ 1:00	95℃ 0:10 60℃ 50℃ 4:00 0:10	60℃ 2：00 4℃ ∞

Table2

Team:Tokyo-NoKoGen/protocols

From 2011.igem.org

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

LB medium (1 L)

LB agar gel (1 L)

Transformation

Inserting plasmid into E. coli

Plasmid extraction

Preparation of plasmid extracted from E. coli

Restriction enzyme digestion of DNA

Cleavage of insert DNA from plasmid

Confirmation and separation of digested DNA

Preparation of agar gel

Agar gel electrophoresis

PCR

Gel purification

Purification of DNA from agar gel

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

Ligation

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

Colony PCR

Sequence analysis

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