View source for Template:Team:Groningen/CONTENT/EXPERIMENTS/PCR ferritin construct (fr1)

{{Team:Groningen/TEMPLATES/LOG
	|title=00:00, 31 Augustus 2015 - 00:00, 31 Augustus 2015
	|executor=Harm Ruesink
	|content=<div class="text">Ferritin was ordered with sequence with prefix and suffix primers from IDT. PCR was performed (figure 1) using NEB Q5 High-Fidelity 2X Master Mix with the prefix and suffix primers.</div>
<div class="object data" id="tbl1">
	<div class="wrapper">
	<div class="header">
		<div class="field fw3”>Primer</div>
		<div class="field fwx">Sequence</div>
	</div>
<div class="record">
	<div class="field fw2">prefix</div>
	<div class="field fwx">CAGGCAGTTGAATTCGCGGCCGCTTCTAGA</div>
	</div>
<div class="record">
	<div class="field fw2">suffix</div>
	<div class="field fwx">CTTGAGCTCCTGCAGCGGCCGCTACTAGTA</div>
	</div>
</div>

<div class="caption">Primers for ferritin PCR.</div>
</div>



<div class="text">The following PCR mix was prepared.</div>
<div class="object data" id="tbl1">
	<div class="wrapper">
	<div class="header">
		<div class="field fw4">Compound</div>
		<div class="field fw3">Amount</div>
			</div>

	<div class="record">
		<div class="field fw4">Q5 polymerase</div>
		<div class="field fw3">9 µL</div>
			</div>

	<div class="record">
		<div class="field fw4">Template DNA (Ferritin IDT)</div>
		<div class="field fw3">1 µL</div>
			</div>

	<div class="record">
		<div class="field fw4">Each primer</div>
		<div class="field fw3">0.3 µL</div>
			</div>

<div class="record">
		<div class="field fw4">\( \mathrm{H_2O}\)</div>
		<div class="field fw3">9 µL</div>
			</div>

	</div>
	<div class="caption">PCR components.</div>

<div class="text">The following thermocycle was used for the PCR.</div>
<div class="object data" id="tbl1">
	<div class="wrapper">
	<div class="header">
		<div class="field fw1">#</div>
		<div class="field fw3">Step</div>
		<div class="field fw3">Temperature</div>
		<div class="field fw3">Time</div>
			</div>
	
	<div class="record">
		<div class="field fw1">1</div>
		<div class="field fw3">Initial denaturation</div>
		<div class="field fw3">98 °C</div>
		<div class="field fw3">5:00</div>
			</div>
	
	<div class="record">
		<div class="field fw1">2</div>
		<div class="field fw3">Denaturation</div>
		<div class="field fw3">98 °C</div>
		<div class="field fw3">0:30</div>
			</div>

	<div class="record">
		<div class="field fw1">3</div>
		<div class="field fw3">Annealing</div>
		<div class="field fw3">60 °C</div>
		<div class="field fw3">0:30</div>
			</div>

	<div class="record">
		<div class="field fw1">4</div>
		<div class="field fw3">Extension</div>
		<div class="field fw3">72 °C</div>
		<div class="field fw3">1:30</div>
			</div>

	<div class="record">
		<div class="field fw1">5</div>
		<div class="field fw3">Go back to step 2 (30x)</div>
		<div class="field fw3"></div>
		<div class="field fw3"></div>
			</div>

	<div class="record">
		<div class="field fw1">6</div>
		<div class="field fw3">Final extension</div>
		<div class="field fw3">72 °C</div>
		<div class="field fw3">10:00</div>
			</div>	
</div>
	<div class="caption">PCR Thermocycle.</div>

<div class="text">The PCR prduct was loaded on a 1% agarose gel with DNA stain G (1:50000) and run for 1 hour on 100 V.</div>

<div class="text">Sample:</div>
<div class="listing">
	<div class="item">1 µL DNA (ferritin).</div>
	<div class="item">1 µL 6x buffer.</div>
        <div class="item">4 µL \( \mathrm{H_2O}\).</div>
</div>
<div class="text">Ladder:</div>
<div class="listing">
	<div class="item">2 µL Thermo Scientific GeneRuler 1kb DNA ladder, ready to use.</div>
</div>

<div class="text">Band of approximately 1600bp was visible on the gel. This corresponds to the size of ferritin.</div>

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