Team:Tokyo-NoKoGen/Protocol
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<h1 id="index_title"></h1> | <h1 id="index_title"></h1> | ||
<ul id="contents"> | <ul id="contents"> | ||
- | <a href="# | + | <a href="#LB"><li><strong>LB medium and agar gel for <i>E. coli</i> </a></strong></li></a> |
- | <a href="# | + | <a href="#transformation"><li><strong>Transformation<strong></li></a> |
- | + | <a href="#plasmid"><li><strong>Plasmid extraction</strong></li></a> | |
- | + | <a href="#restriction"><li><strong>Restriction Enzyme digestion</strong></li> | |
+ | <a href="#agar"><li><strong>Agar gel electrophoresis</strong></li> | ||
+ | <a href="#pcr"><li><strong>PCR</strong></li> | ||
+ | <a href="#restriction"><li><strong>Restriction Enzyme digestion</strong></li> | ||
+ | <a href="#restriction"><li><strong>Restriction Enzyme digestion</strong></li> | ||
+ | <a href="#restriction"><li><strong>Restriction Enzyme digestion</strong></li> | ||
+ | <a href="#restriction"><li><strong>Restriction Enzyme digestion</strong></li> | ||
<li><strong></strong></li> | <li><strong></strong></li> | ||
<li><strong></strong></li> | <li><strong></strong></li> | ||
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<div id="main"> | <div id="main"> | ||
- | Protocols</font> | + | <font size=7>Protocols</font> |
- | + | <BR> | |
- | < | + | <BR> |
+ | <h1 id="LB"> | ||
LB medium and LB agar gel | LB medium and LB agar gel | ||
Medium for cultivation of E. coli | Medium for cultivation of E. coli | ||
- | </ | + | </h1> |
<h3>LB medium (1 L)</h3> | <h3>LB medium (1 L)</h3> | ||
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</table><br><br> | </table><br><br> | ||
- | < | + | <h1 id="transformation">>Transformation</h1> |
<h3>Inserting plasmid into <i>E. coli</i></h3> | <h3>Inserting plasmid into <i>E. coli</i></h3> | ||
1; Incubate frozen competent cell (DH5α) on the ice for a few minutes. <br> | 1; Incubate frozen competent cell (DH5α) on the ice for a few minutes. <br> | ||
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6; Spread culture medium on LB agar plate with appropriate antibiotic.<br><br> | 6; Spread culture medium on LB agar plate with appropriate antibiotic.<br><br> | ||
- | < | + | <h1 id="plasmid">Plasmid extraction</h1> |
<h3>Preparation of plasmid extracted from <i>E. coli</i></h3> | <h3>Preparation of plasmid extracted from <i>E. coli</i></h3> | ||
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19; 40 μL of supernatant into new 500 μL tube.<br><br> | 19; 40 μL of supernatant into new 500 μL tube.<br><br> | ||
- | < | + | <h1 id="restriction">Restriction enzyme digestion of DNA</h1> |
<h3>Cleavage of insert DNA from plasmid</h3> | <h3>Cleavage of insert DNA from plasmid</h3> | ||
1; Mix DNA and restriction enzyme (Table).<br> | 1; Mix DNA and restriction enzyme (Table).<br> | ||
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5; Store gel in 1×TAE.<br><br><br> | 5; Store gel in 1×TAE.<br><br><br> | ||
- | < | + | <h1 id=electro>Agar gel electrophoresis</h1> |
1; Place agar gel and pour 1×TAE in electrophoresis chamber.<br> | 1; Place agar gel and pour 1×TAE in electrophoresis chamber.<br> | ||
2; Load DNA ladder and DNA sample mixed with loading dye on agar gel.<br> | 2; Load DNA ladder and DNA sample mixed with loading dye on agar gel.<br> | ||
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6; Confirm the length of digested DNA.<br><br> | 6; Confirm the length of digested DNA.<br><br> | ||
- | < | + | <h1 id=pcr>PCR</h1> |
1; Add 25 μL of reagent solution (Table 1) to PCR tube.<br> | 1; Add 25 μL of reagent solution (Table 1) to PCR tube.<br> | ||
2; Amplify target DNA with PCR program (Table 2).<br> | 2; Amplify target DNA with PCR program (Table 2).<br> | ||
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- | < | + | <h1 id=gel>Gel purification</h1> |
<h3>Purification of DNA from agar gel</h3> | <h3>Purification of DNA from agar gel</h3> | ||
<h4>GENECLEAN® II Kit(NaI、glass milk、NEW Wash)/Qbiogene</h4> | <h4>GENECLEAN® II Kit(NaI、glass milk、NEW Wash)/Qbiogene</h4> | ||
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14. Transfer supernatant including objective DNA into new tube.<br><br> | 14. Transfer supernatant including objective DNA into new tube.<br><br> | ||
- | < | + | <h1 id=ligation>Ligation</h1><br> |
<h4>Ligation inset DNA and vector<br> | <h4>Ligation inset DNA and vector<br> | ||
DNA Ligation kit Ver 2.1(SolutionⅠ)/Takara<br></h4> | DNA Ligation kit Ver 2.1(SolutionⅠ)/Takara<br></h4> | ||
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<br><br> | <br><br> | ||
- | < | + | <h1 id=colony>Colony PCR</h1><br> |
Confirmation of insert DNA in plasmid, directly doing PCR on <i>E. coli</i><br> | Confirmation of insert DNA in plasmid, directly doing PCR on <i>E. coli</i><br> | ||
1; Add 10 μL of reagent solution (Table 1) to PCR tube.<br> | 1; Add 10 μL of reagent solution (Table 1) to PCR tube.<br> | ||
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Table2<br><br> | Table2<br><br> | ||
- | < | + | <h1 id=sequence>Sequence analysis</h1> |
Identification of insert DNA<br> | Identification of insert DNA<br> | ||
*Preparation of PCR product<br> | *Preparation of PCR product<br> |
Revision as of 04:29, 27 September 2013
Protocols
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.
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.
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.
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.
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.
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.
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.
2; Amplify target DNA with PCR program (Table 2).
3; Confirm the band of DNA by agar gel electrophoresis.
Table 1
Table2
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 inset DNA and vector
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.
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.
Table 1
Table2
*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.
Table 1
Table2
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 |
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℃ ∞ |
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 |
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