Team:UB Indonesia/backup

From 2014.igem.org

(Difference between revisions)
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<b>Ligation of sample and PSB1C3</b>
<b>Ligation of sample and PSB1C3</b>
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Make the composisition of the mixture, include:
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<ul type="circle">
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<li>10x KAPA buffer 2 μl</li>
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<li>Add (5 u/ μl) 1 μl </li>
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<li>Add Psb1c3 (25 ng/ μl) 4 μl </li>
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<li>Add sample 4 μl</li>
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<li>Add ddH2O and then mix gentle</li>
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<li>Ligate at 16˚C for 30 minutes, then heat kill 80˚C for 20 minutes</li>
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<li>Transform with 2 ul of product</li>
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</ul><br>
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<b>Transformation</b>
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<ul type="circle">
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<li>Thaw competent cells on ice.</li>
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<li>Place 25 µL of cells in a pre-chilled 2ml tube </li>
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<li>Place 25µL in 2ml tube and label it for another. Use it for control. </li>
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<li>Add 150 µL of resuspended DNA to 2ml tube. </li>
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<li>Pipet up and down gently for a few times. keep the competent cells still on ice.</li>
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<li>Add 1 µL of the RFP Control/li>
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<li>Incubate the cells on ice for30 minutes.</li>
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<li>Heat shock at 42ºC for 60 seconds.</li>
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<li>Return to ice for 5 minutes. </li>
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<li>Add 250 μl of SOC media to each transformation </li>
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<li>Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Note:2 hour recovery time helps in transformation efficiency </li>
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<li>Do not forget to label two petri dishes with selective LB agar and kanamycin antibiotic with antibiotic resistance, part number and plasmid backbone. </li>
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<li>Place 20 µl and 200 µl of the transformation to dishes. Spread it. This step help ensure that you will be able to pick out a single colony.</li>
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<li>Place 20 µl and 200 µl of the transformation to the dishes. Spread it.</li>
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<li>Incubate the plates at 37ºC for 12-14 hours </li>
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<li>Count the colonies on the 20 μl control plate</li>
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<li>Calculated the competent cell.</li>
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</ul><br>
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<b>Polymerase Chain Reaction</b><br>
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<ul>
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<li>Make the composisition of the mixture, include:</li>
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Add 23 μl of ddH20 <br>
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Add 0.5  μl of Primer Forward <br>
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Add 0.5 μl of Primer Reverse <br>
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Add 1 μl of DNA samples. There should be a volume of 25 ul <br>
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<li>Running on PCR machinewith 35x PCR cycle, include :</li>
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Hot start : 5 minutes on 94oC<br>
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Denaturation : 30 second on 94oC<br>
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Annealing : 30 second on 50oC<br>
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Extention : 60 second on 72oC<br>
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Post extention : 10 minutes on 72oC<br>
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</ul>
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<b>Annealing Oligonucleotide</b>
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<ul>
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<li>siRNA forward (10 μl) and siRNA reverse (10 μl) mix gentle</li>
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<li>Add TE buffer 25 μl, ddH2O 25 μl</li>
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<li>Heat to 95oC for 2 minutes</li>
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<li>Ramp cool to 25oC over period of 45 minutes</li>
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</ul>
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 +
<b>Agarose Gel Electrophoresis</b><br>
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<ul>
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<li>Dissolve the 2% agar, 0,3 gr agarose on 15 ml TBE + 1 μl EtBr.</li>
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<li>Heat the mixture until the agar is dissolve.</li>
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<li>Cool the mixture and the comb on the plate.</li>
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<li>Pour the cooled agar in to the plate.</li>
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<li>When the agar has solidified, removed the come carefully.</li>
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<li>Places the gel in to electrophoresis chamber.</li>
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<li>Pour 1X TBE over gel so that gel is covered by a 3-5mm buffer.</li>
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<li>Load samples in the wells in the gels : 1 μl DNA and 3 μl loading dye.</li>
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<li>Load DNA ladder 2 μl to the well.</li>
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<li>Place the lid on the chamber and connect the electrode leads to the power supply.</li>
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<li>Turn on the power supply and adjust the voltage 50-100 volts.</li>
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<li>Run the gel for 5-10 minutes.</li>
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<li>Load samples into well.</li>
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<li>Hook electrodes to gel apparatus.</li>
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<li>Run the apparatus at 100V for 20 minutes.</li>
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<li>Visualize the gel and record the results by UV-transilluminator</li>
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</ul>
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 +
<b>Isolation Plasmid used Kit-free Alkaline Lysis Plasmid Miniprep</b><br>
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<ul>
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<li>Prepare the following solutions:</li>
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a. Solution I (Resuspension buffer)<br>
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    i. 25 mM Tris-HCl (pH 8) <br>
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    ii. 50 mM glucose <br>
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    iii. 10 mM EDTA<br>
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b. Solution II (Denaturing Solution) <br>
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    i. 0.2 N NaOH <br>
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    ii. 1.0% SDS <br>
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c. Solution III (Renaturing Solution: Potassium Acetate) <br>
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    i. 120 mL 5M Potassium acetate <br>
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    ii. 23 mL glacial acetic acid <br>
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    iii. 57 mL of de-ionized water<br>
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Store Solution I at 4°C <br>
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Store Solution II at room temperature <br>
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Store Solution III at 4°C<br>
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<li>Grow 2mL overnight cultures from single colonies of bacteria containing your plasmid of interest.</li>
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<li> Add 1.5mL of the stock culture to a 1.75mL microfuge tube.</li>
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<li>Centrifuge in microfuge tube at 10,000g for 30sec.</li>
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<li>Pour off the supernatant, being careful not to disturb the bacterial pellet.</li>
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<li>Resuspend the pellet in 100ul of cold Solution I.</li>
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<li>Vortex the solution for 2 minutes or until all bacteria are fully resuspended.</li>
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<li>Add 200μl of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured.</li>
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<li>Incubate solution on ice for 5 minutes.</li>
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<li>Add 150μl of cold Solution III to each tube.</li>
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<li>Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.</li>
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<li>Incubate tube on ice for 5 minutes.</li>
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<li>Centrifuge the tube for 5 minutes at 12,000g</li>
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<li>Add either 700μL of cold 100% ethanol or 350uL room temperature isopropanol to the solution to precipitate the plasmid DNA.</li>
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<li>Pour out the supernatant</li>
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<li>Air dry the pellet (can be done by inverting the tube at an angle over kimwipe) for 20-30 minutes.</li>
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<li>Resuspend pellet with 25-50μl of TE.</li>
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</ul><br><br>
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<b>GATE kit For Easy TAL Effector Assembly</b>
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<ul><li>Choose your DNA target sequence. </li></ul>
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This sequence should be 14 bp long and they start and end with a thymine base.<br>
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1. Find the right di-repeats: <br>
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2. Remove the flanking thymine bases from your target sequence, split it into six pairs of nucleotides and number the repeats from one to six.To get the needed concentration of 60ng/μl please resuspend the DNA in 10μl H2O<br>
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</p>
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<center>TGCAAGTTCGAGCT<br>
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GC-AA-GT-TC-GA-GC<br>
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1. GC 2. AA 3. GT 4. TC 5. GA 6.GC<br>
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</center>
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<p align="justify">
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3. Insert the di-repeats into the expression vector. <br>
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Mix the following components in a PCR tube and  prepare a negative control lacking the di-repeats.<br>
</p>
</p>
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<center>
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<table><tr><th>Component</th><th>Amount (μl)</th></tr>
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<tr><td>6 di-repeat plasmids (60ng)<br>
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Expression vector (170ng)<br>
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(pTALEN/pTAL-TF/pTAL-KRAB)<br>
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T4 Ligase Buffer (10x)<br>
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T4 Ligase (60 U)<br>
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BsmBI (15 U)<br>
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dH2O</td>
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<td>
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1 each<br>
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1<br>
 +
<br>
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<br>
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2<br>
 +
1<br>
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1,5<br>
 +
8,5<br>
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</td></tr>
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<tr><td>Total</td><td>20</td></tr>
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</table>
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</center>
                                         </div>
                                         </div>
<h2 class="font-thin"><font color="#fff">Protocols</font></h2>
<h2 class="font-thin"><font color="#fff">Protocols</font></h2>

Revision as of 17:14, 16 October 2014

iGEM2014 | UB INDONESIA

BRAWIJAYA UNIVERSITY , INDONESIA

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