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| - | <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/HeatShock">PCR </a></span> </regulartext> | + | <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols">PCR </a></span> </regulartext> |
| | </div> | | </div> |
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| - | <b> Isolation of mRNA from <i>Helianthus tuberosus</i> </b> | + | <b> Polymerase Chain Reaction </i> </b> |
| | <p> | | <p> |
| - | (The procedure we used for mRNA isolation, is a modified version of the Qiagen RNeasy protocol for plant mRNA isolation.)
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| - | <p>
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| - | Small tubers from Helianthus tuberosus were washed with untreated water and cut in rough slices with an everyday boxcutter.</b>
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| - | The slices were distributed in cryotubes and weighed, the weights were noted down, and submerged in liquid nitrogen(flash-freeze at -196 °C). Flash-freezing is necessary to halt all RNase activity. </b>
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| - | RLT buffer is mixed at this point, for later use. For each mL RLT, add uL beta-mercaptoethanol. Do this in a fume cabinet, as mercaptoethanol smells really bad and is toxic!. </b>
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| - | After 20 minutes 600mg is extracted from the cryotubes, the rest is stored for later use. The 600mg plant material is grinded to a fine dust using a mortar. It is important to keep the plant material frozen during the grinding, to keep the cell wall rigid enough to destroy it.</b>
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| - | The plant powder is transferred to a tube and dissolved in RLT buffer(450uL buffer op 100mg plant material). We used approx. 3mL for 600mg material.</b>
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| - | The solution is mixed using a vortex mixer.</b>
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| - | 500 uL of the solution is transfered to a 2mL treated with ultrasound to homogenize the content and further disrupt the cell wall. 500 uL is transfered to a 2mL tube as a control.</b>
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| - | The samples are centrifuged at 14.500 rpm for 60 seconds to pellet out large organelles.
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| - | 0,5 vol. 96-100% ethanol is added(250uL) to the sample, mixed by pipette. </b>
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| - | Transfer the samples to spin-column(Qiagen RNeasy mini kit, for RNA isolation from animal cells) in a 2mL sampletube and do the following:
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| - | <p>
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| - | -spin at 10.000rmp for 15s, discard flowthrough
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| - | add 700uL RW1 til spin column </b>
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| - | -spin at 10.000rmp for 15s, discard flowthrough
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| - | add 500uL RPE buffer til spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol)</b>
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| - | -spin at 10.000rmp for 15s, discard flowthrough
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| - | add another 500uL RPE buffer to the spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol)</b>
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| - | -spin at 10.000rmp for 15s, discard flowthrough
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| - | Transfer the columns to new 2mL centrifuge tubes. Centrifuge at full speed for 1 min.
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| - | Transfer spin columns to new 1.5mL sample tubes</b>
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| - | -add 30-50uL RNase-free water</b>
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| - | -spin at 10.000rpm for 60 seconds</b>
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| - | Repeat the prior step once.
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| - | <p>
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| - | The mRNA is now isolated in the 60-100uL at the bottom of the collection tubes
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| - | <p>
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| - | The solutions were tested on <b>Nanodrop</b>:
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| - | <p>
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| - | Control: 627 ng/uL </br>
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| - | Sonic: 747 ng/uL
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| - | <p>
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| - | As expected, disruption of the cell wall by ultrasound resulted in a higher yield.
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