Team:Tokyo-NoKoGen/protocols
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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. coliLB 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.
TransformationInserting plasmid into E. coli1; 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 extractionPreparation of plasmid extracted from E. coli1; 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 DNACleavage of insert DNA from plasmid1; 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 DNAPreparation of agar gel1; 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 electrophoresis1; 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. PCR1; 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.
Gel purificationPurification of DNA from agar gelGENECLEAN® II Kit(NaI、glass milk、NEW Wash)/Qbiogene1; 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. LigationLigation inset DNA and vector
1; Mix the insert DNA, vector and solution I (Table). |
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 |
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℃ ∞ |
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
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