Team:UB Indonesia/backup
From 2014.igem.org
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<b>Ligation of sample and PSB1C3</b> | <b>Ligation of sample and PSB1C3</b> | ||
+ | Make the composisition of the mixture, include: | ||
+ | <ul type="circle"> | ||
+ | <li>10x KAPA buffer 2 μl</li> | ||
+ | <li>Add (5 u/ μl) 1 μl </li> | ||
+ | <li>Add Psb1c3 (25 ng/ μl) 4 μl </li> | ||
+ | <li>Add sample 4 μl</li> | ||
+ | <li>Add ddH2O and then mix gentle</li> | ||
+ | <li>Ligate at 16˚C for 30 minutes, then heat kill 80˚C for 20 minutes</li> | ||
+ | <li>Transform with 2 ul of product</li> | ||
+ | </ul><br> | ||
+ | <b>Transformation</b> | ||
+ | <ul type="circle"> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Place 25 µL of cells in a pre-chilled 2ml tube </li> | ||
+ | <li>Place 25µL in 2ml tube and label it for another. Use it for control. </li> | ||
+ | <li>Add 150 µL of resuspended DNA to 2ml tube. </li> | ||
+ | <li>Pipet up and down gently for a few times. keep the competent cells still on ice.</li> | ||
+ | <li>Add 1 µL of the RFP Control/li> | ||
+ | <li>Incubate the cells on ice for30 minutes.</li> | ||
+ | <li>Heat shock at 42ºC for 60 seconds.</li> | ||
+ | <li>Return to ice for 5 minutes. </li> | ||
+ | <li>Add 250 μl of SOC media to each transformation </li> | ||
+ | <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> | ||
+ | <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> | ||
+ | <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> | ||
+ | <li>Place 20 µl and 200 µl of the transformation to the dishes. Spread it.</li> | ||
+ | <li>Incubate the plates at 37ºC for 12-14 hours </li> | ||
+ | <li>Count the colonies on the 20 μl control plate</li> | ||
+ | <li>Calculated the competent cell.</li> | ||
+ | </ul><br> | ||
+ | |||
+ | <b>Polymerase Chain Reaction</b><br> | ||
+ | <ul> | ||
+ | <li>Make the composisition of the mixture, include:</li> | ||
+ | Add 23 μl of ddH20 <br> | ||
+ | Add 0.5 μl of Primer Forward <br> | ||
+ | Add 0.5 μl of Primer Reverse <br> | ||
+ | Add 1 μl of DNA samples. There should be a volume of 25 ul <br> | ||
+ | <li>Running on PCR machinewith 35x PCR cycle, include :</li> | ||
+ | Hot start : 5 minutes on 94oC<br> | ||
+ | Denaturation : 30 second on 94oC<br> | ||
+ | Annealing : 30 second on 50oC<br> | ||
+ | Extention : 60 second on 72oC<br> | ||
+ | Post extention : 10 minutes on 72oC<br> | ||
+ | </ul> | ||
+ | |||
+ | <b>Annealing Oligonucleotide</b> | ||
+ | <ul> | ||
+ | <li>siRNA forward (10 μl) and siRNA reverse (10 μl) mix gentle</li> | ||
+ | <li>Add TE buffer 25 μl, ddH2O 25 μl</li> | ||
+ | <li>Heat to 95oC for 2 minutes</li> | ||
+ | <li>Ramp cool to 25oC over period of 45 minutes</li> | ||
+ | </ul> | ||
+ | |||
+ | <b>Agarose Gel Electrophoresis</b><br> | ||
+ | <ul> | ||
+ | <li>Dissolve the 2% agar, 0,3 gr agarose on 15 ml TBE + 1 μl EtBr.</li> | ||
+ | <li>Heat the mixture until the agar is dissolve.</li> | ||
+ | <li>Cool the mixture and the comb on the plate.</li> | ||
+ | <li>Pour the cooled agar in to the plate.</li> | ||
+ | <li>When the agar has solidified, removed the come carefully.</li> | ||
+ | <li>Places the gel in to electrophoresis chamber.</li> | ||
+ | <li>Pour 1X TBE over gel so that gel is covered by a 3-5mm buffer.</li> | ||
+ | <li>Load samples in the wells in the gels : 1 μl DNA and 3 μl loading dye.</li> | ||
+ | <li>Load DNA ladder 2 μl to the well.</li> | ||
+ | <li>Place the lid on the chamber and connect the electrode leads to the power supply.</li> | ||
+ | <li>Turn on the power supply and adjust the voltage 50-100 volts.</li> | ||
+ | <li>Run the gel for 5-10 minutes.</li> | ||
+ | <li>Load samples into well.</li> | ||
+ | <li>Hook electrodes to gel apparatus.</li> | ||
+ | <li>Run the apparatus at 100V for 20 minutes.</li> | ||
+ | <li>Visualize the gel and record the results by UV-transilluminator</li> | ||
+ | </ul> | ||
+ | |||
+ | <b>Isolation Plasmid used Kit-free Alkaline Lysis Plasmid Miniprep</b><br> | ||
+ | <ul> | ||
+ | <li>Prepare the following solutions:</li> | ||
+ | a. Solution I (Resuspension buffer)<br> | ||
+ | i. 25 mM Tris-HCl (pH 8) <br> | ||
+ | ii. 50 mM glucose <br> | ||
+ | iii. 10 mM EDTA<br> | ||
+ | b. Solution II (Denaturing Solution) <br> | ||
+ | i. 0.2 N NaOH <br> | ||
+ | ii. 1.0% SDS <br> | ||
+ | c. Solution III (Renaturing Solution: Potassium Acetate) <br> | ||
+ | i. 120 mL 5M Potassium acetate <br> | ||
+ | ii. 23 mL glacial acetic acid <br> | ||
+ | iii. 57 mL of de-ionized water<br> | ||
+ | Store Solution I at 4°C <br> | ||
+ | Store Solution II at room temperature <br> | ||
+ | Store Solution III at 4°C<br> | ||
+ | |||
+ | <li>Grow 2mL overnight cultures from single colonies of bacteria containing your plasmid of interest.</li> | ||
+ | <li> Add 1.5mL of the stock culture to a 1.75mL microfuge tube.</li> | ||
+ | <li>Centrifuge in microfuge tube at 10,000g for 30sec.</li> | ||
+ | <li>Pour off the supernatant, being careful not to disturb the bacterial pellet.</li> | ||
+ | <li>Resuspend the pellet in 100ul of cold Solution I.</li> | ||
+ | <li>Vortex the solution for 2 minutes or until all bacteria are fully resuspended.</li> | ||
+ | <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> | ||
+ | <li>Incubate solution on ice for 5 minutes.</li> | ||
+ | <li>Add 150μl of cold Solution III to each tube.</li> | ||
+ | <li>Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.</li> | ||
+ | <li>Incubate tube on ice for 5 minutes.</li> | ||
+ | <li>Centrifuge the tube for 5 minutes at 12,000g</li> | ||
+ | <li>Add either 700μL of cold 100% ethanol or 350uL room temperature isopropanol to the solution to precipitate the plasmid DNA.</li> | ||
+ | <li>Pour out the supernatant</li> | ||
+ | <li>Air dry the pellet (can be done by inverting the tube at an angle over kimwipe) for 20-30 minutes.</li> | ||
+ | <li>Resuspend pellet with 25-50μl of TE.</li> | ||
+ | </ul><br><br> | ||
+ | |||
+ | <b>GATE kit For Easy TAL Effector Assembly</b> | ||
+ | <ul><li>Choose your DNA target sequence. </li></ul> | ||
+ | This sequence should be 14 bp long and they start and end with a thymine base.<br> | ||
+ | 1. Find the right di-repeats: <br> | ||
+ | 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> | ||
+ | </p> | ||
+ | <center>TGCAAGTTCGAGCT<br> | ||
+ | GC-AA-GT-TC-GA-GC<br> | ||
+ | 1. GC 2. AA 3. GT 4. TC 5. GA 6.GC<br> | ||
+ | </center> | ||
+ | <p align="justify"> | ||
+ | 3. Insert the di-repeats into the expression vector. <br> | ||
+ | Mix the following components in a PCR tube and prepare a negative control lacking the di-repeats.<br> | ||
</p> | </p> | ||
+ | <center> | ||
+ | <table><tr><th>Component</th><th>Amount (μl)</th></tr> | ||
+ | <tr><td>6 di-repeat plasmids (60ng)<br> | ||
+ | Expression vector (170ng)<br> | ||
+ | (pTALEN/pTAL-TF/pTAL-KRAB)<br> | ||
+ | T4 Ligase Buffer (10x)<br> | ||
+ | T4 Ligase (60 U)<br> | ||
+ | BsmBI (15 U)<br> | ||
+ | dH2O</td> | ||
+ | <td> | ||
+ | 1 each<br> | ||
+ | 1<br> | ||
+ | <br> | ||
+ | <br> | ||
+ | 2<br> | ||
+ | 1<br> | ||
+ | 1,5<br> | ||
+ | 8,5<br> | ||
+ | </td></tr> | ||
+ | <tr><td>Total</td><td>20</td></tr> | ||
+ | </table> | ||
+ | </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