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<p> </p>
<p>(1)</p>
<p> [[File:12.png|600px]]</p>
<p> pRA-GFP-EcoR1-pRL-unknown-mETP-SpyTag-Stop</p>
<p> </p>
<p>-><strong> How to make this cloning?</strong></p>
<p>(1)By Using pRL-EcoR1 forward primer, mETP-SpyTag-Stop-Xho1 primer, make pRL-EcoR1-mETP-SpyTag-Stop-Xho1 by Ex-Tag PCR</p>
<p>(2) Insert template gained from (1) in T-Vector to check whether it is really pRL-EcoR1-mETP-SpyTag-Stop-Xho1 or not.</p>
<p>(3) Use EcoR1, Xho1 Digestion enzyme to double digest T vector</p>
<p>(4) Double Digest pRA GFP vector(empty vector) and purify it.</p>
<p>(5) ligate (3), (4) product</p>
<p> </p>
<p><strong>-> Detailed on Each Steps</strong></p>
<p>(1) Ex-Tag PCR process</p>
<p> </p>
<p>->Template(pRA-GFP, 20ng): 1ul</p>
<p>->Primer: 1,1ul</p>
<p>->dNTP(10nM): 1ul</p>
<p>->10X Ex-Tag Buffer: 2.5ul</p>
<p>-> Ex-Tag polymerase: 1ul</p>
<p>-> D.W: 17.5ul</p>
<p>----------------------------------------</p>
<p>Total: 25ul</p>
<p> </p>
<p>PCR</p>
<p>->Temperature Gradient : 54,56,58</p>
<p>->98 celsius : 2min</p>
<p>->98 celsius : 10sec</p>
<p>->57 celsius : 30sec</p>
<p>->72 celsius : 30sec(insert 300bp)</p>
<p>->72 celsius : 5min</p>
<p>X35</p>
<p> </p>
