Team:Tokyo-NoKoGen/protocols

Tokyo-NokoGen 2011 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology

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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 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

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 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 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.

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

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.

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