Team:Tokyo-NoKoGen/experiments

From 2010.igem.org

(Difference between revisions)

Latest revision as of 23:26, 27 October 2010

We noted "experimental protocols" and "Lab note".
All our experiments were carried out according to the protocol described above.

And we also provided a pdf file of our protocols.
It would help iGEM members to do experiment in hereafter.

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+ We noted <a href="https://2010.igem.org/Team:Tokyo-NoKoGen/experiments/Protocols">"experimental protocols"</a> and <a href="https://2010.igem.org/Team:Tokyo-NoKoGen/experiments/Lab_note">"Lab note".</a><br>
-Protocols</font>
+ All our experiments were carried out according to the protocol described above.<br><br>
-<h2>
+  And we also provided <a href="https://static.igem.org/mediawiki/2010/c/cf/Tokyo-NoKoGen_protcols.pdf">a pdf file</a> of our protocols.<br>
-LB medium and LB agar gel
+ It would help iGEM members to do experiment in hereafter.
-Medium for cultivation of E. coli
-</h2>
-<h3>LB medium (1 L)</h3>
-; Add about 900 mL of distilled water to beaker.<br>
-; Add reagents (Table) and stir. <br>
-; Add distilled water up to 1 L and take LB medium to media bottle.<br>
-; Autoclave for 20 min at 120°C.<br>
-<h3>LB agar gel (1 L)</h3>
-; Prepare LB medium without autoclave (Steps 1-3 of 1L scale of LB medium).<br>
-; Add 15 g of agar and stirrer bar.<br>
-; Autoclave for 20 minutes at 120°C.<br>
-; Stir and cool LB medium with agar, add appropriate antibiotic (50 μL).<br>
-; Pour LB medium (Step 4) in plate and cool down in clean bench.<br>
-<h2>Transformation</h2>
-<h3>Preparation of E. coli contain particular plasmid</h3>
-; Incubate frozen competent cell (DH5α) on the ice for a few minutes. <br>
-; Add 1~5 μL of plasmid to competent cell (DH5α) on the ice.<br>
-; Incubate for 20 – 30 minutes on the ice.<br>
-; Incubate for 45 seconds at 42°C.<br>
-; Add 1 mL LB medium and cultivate for 1 hour at 37°C.<br>
-; Spread culture medium on LB agar plate with appropriate antibiotic.<br>
-<h2>Plasmid extraction</h2>
-<h3>Preparation of plasmid extracted from <i>E. coli</i></h3>
-; 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>
-; Move the culture medium to 1.5 mL tube.<br>
-; Centrifuge for 5 seconds at 15,000×g and 4 °C and discard supernatant.<br>
-; Add 100 μL Solution 1 to the pellet and resuspend and incubate for 3 minutes.<br>
-; Add 100 μL Solution 2, invert tube gently 5 times and incubate for 3 minutes.<br>
-; Add 100 μL Solution 3, invert tube 5 times and incubate for 3 minutes. <br>
-; Add 200 μL Solution 4 and invert tube 5 times and centrifuge for 3 minutes at 15,000×g and 4 °C.<br>
-; Take supernatant to new 1.5 mL tube and centrifuge for 3 minutes at 15,000×g and 4 °C.<br>
-; Vortex Bind mix for 1 min and add 800 μL Bind mix to new 1.5 mL tube.<br>
-; Add 400 μL supernatant after centrifugation (Step 10) to tube containing Bind mix (Step 11) and mix.<br>
-; Incubate for 3 minutes, centrifuge for 3 seconds at 5,000×g and 4 °C, and discard supernatant.<br>
-; Add 1 mL of 50% ethanol and resuspend. <br>
-; Centrifuge for 3 seconds at 5,000×g and 4 °C and discard supernatant.<br>
-; Repeat wash (Steps 14-15).<br>
-; Dry pellet for a few minuet under a vacuum to remove residual ethanol.<br>
-; Add 50 μL nuclease-free water or TE buffer and incubate for 3 minutes at 65°C.<br>
-; Centrifuge for 3 minutes at 15,000×g and 4 °C.<br>
-; 40 μL of supernatant into new 500 μL tube.<br>
-<h2>Restriction enzyme digestion of DNA</h2>
-<h3>Cleavage of insert DNA from plasmid</h2>
-; Mix DNA and restriction enzyme (Table).<br>
-; Incubate for 2 hours at 37°C.<br>
-; Incubate for 10 minutes at 65°C.<br>
-; Confirm the band of DNA by agar gel electrophoresis.<br>
-<h2Agar gel electrophoresis</h2>
-<h3>Confirmation and separation of digested DNA</h2>
-<h4>Preparation of agar gel</h4>
-; Add 1 g of agar to 100 mL of 1×TAE.<br>
-; Boil and stir until solution is clear.<br>
-; Cool down, pour to gel form and set gel corm.<br>
-; Incubate until gel dry out.<br>
-; Stare gel in 1×TAE.<br>
-<h2>Agar gel electrophoresis</h2>
-; Place agar gel and pour 1×TAE in electrophoresis chamber.<br>
-; Load DNA ladder and DNA sample mixed with loading dye on agar gel.<br>
-; Electrophorese for 20 minutes at 100 V.<br>
-; Stain gel by Sybr SafeTM Gel Stain (Invitrogen).<br>
-; Visualize the band of DNA using UV light.<br>
-; Confirm the length of digested DNA.<br>
-<h2>Gel purification</h2>
-<h3>Purification of DNA from agar gel</h3>
-<h4>GENECLEAN® II Kit(NaI、glass milk、NEW Wash)/Qbiogene</h4>
-; Cut the objective band in the agar gel after electrophoresis and stain with SYBR Safe.<br>
-; Put the gel including objective DNA into 1.5 mL tube and measure the mass of that.<br>
-; Add 2.5-3 fold volume NaI solution into the tube (Gel: NaI =1 mg : 1 μL ).<br>
-; Incubate the gel at 50°C for 5 minute.<br>
-; Add 10 μL of glass milk and vortex.<br>
-; Incubate for 5 minutes and vortex per a minute.<br>
-; Centrifuge for 5 seconds at 15,000×g and 4°C and discard the supernatant.<br>
-; Add the 500μL of New Wash and resuspend.<br>
-; Centrifuge for 5 seconds at 15,000×g and 4°C.<br>
-; Repeat wash (Steps 8-9).<br>
-; Dry the pellet for 5-10 minutes under vacuum.<br>
-. Add 20　μL of nuclease-free water and resuspend.<br>
-. Centrifuge for 5 seconds at 15,000×g and 25°C.<br>
-. Transfer supernatant including objective DNA into new tube.<br>
-<h3>Ligation</h3><br>
-<h4>Ligation inset DNA and vector<br>
-DNA Ligation kit Ver 2.1(SolutionⅠ)/Takara<br></h4>
-; Mix the insert DNA, vector and solution I (Table).<br>
-; Incubation at 16°C for 30 minute.<br>
-; Transform E. coli with ligation sample.<br>
-<h3>Colony PCR</h3><br>
-Confirm of insert DNA in plasmid, directly using E. coli at PCR.<br>
-; Add 10 μL of reagent solution (Table 1) to PCR tube.<br>
-; Pick up single colony from agar plate with tooth pick and sting replica plate (new LB agar plate).<br>
-; Put and stir toothpick to reagent solution (Step 1).<br>
-; Amplify insert DNA with PCR program (Table 2).<br>
-; Electrophorese PCR sample with agar gel.<br>
-; Check the band and length of insert DNA and decide the colony with insert DNA.<br>
-<h3>Sequence analysis</h3>
-Identification of insert DNA<br>
-*Preparation of PCR product<br>
-Big Dye® Terminator Cycle Sequencing Kit Ver. 3.1 (Premix, Buffer) / Applied Biosystems<br>
-; Add reagent solution (Table 1) to PCR tube and amplify insert DNA with PCR program<br> (Table2).
-*Purification of PCR product and sequence analysis<br>
-Agencourt CleanSEQ® and 96 R ring Super Magnetic Plate® / Beckman Coulter<br>
-; Add 10 μL of Agencourt CleanSEQ® 10 µL to PCR product.<br>
-; Add 62 μL of 85% ethanol, mix and incubate for 3 minutes.<br>
-; Incubate for 3 minutes on 96 R ring Super Magnetic Plate® and discard supernatant.<br>
-; Add 100 μL of 85% ethanol and mix.<br>
-; Incubate for 3 minutes on 96 R ring Super Magnetic Plate® and discard supernatant.<br>
-; Repeat wash (Steps 4-5).<br>
-; Dry for 10 minutes.<br>
-; Add 40 μL nuclease-free water and mix.<br>
-; Transfer 30μL of clear sample into a new plate for loading on the detector.<br>
-; Load sample on sequencer and analyze.<br>
-</p>
-<h2>
-Time schedule
-</h2>
-<h3>
-<ul>
-<li>June</li>
-<li>July</li>
-<li>August</li>
-<li>September</li>
-<li>October</li>
-<li>November</li>
-</ul>
-</h3>
-<h2>
-Progress in this year
-</h2>
-<h2>
-Future plan
-</h2>
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