https://eln.excellgen.com/index.php?action=history&feed=atom&title=1_Step_Gateway_Cloning 2026-10-18T02:14:31Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1_Step_Gateway_Cloning&diff=10094&oldid=prev 2013-08-21T13:11:36Z

<p></p> <p><b>New page</b></p><div>&#039;&#039;&#039;Rapid one-step LR recombinational cloning using an enzyme mixture containing Int, IHF and Xis (Gateway Clonase) and two step PCR amplification&#039;&#039;&#039; <br /> <br /> Success rate 80% <br /> <br /> == Gene specific primers: ==<br /> <br /> &#039;&#039;&#039;GSS-F:&#039;&#039;&#039;&lt;br&gt; SbfI Kozak GSS&lt;br&gt;AGGCT cctgcagg CCACCATG NNNNNNNNNNNNNNNNNNNNN <br /> <br /> &#039;&#039;&#039;GSS-R:&#039;&#039;&#039;&lt;br&gt; GSS * Xho1 &lt;br&gt;(NNNNNNNNNNNNNNNNNNNNN TAG ctcgag ACCCAGCTTTC) <br /> <br /> For example: GAAAGCTGGGT ctcgag CTA N(15–25) <br /> <br /> == Universal Primers ==<br /> <br /> &#039;&#039;&#039;attL1-F&#039;&#039;&#039;&lt;br&gt;ccccGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCA GGCTcctgcaggCCACCATG <br /> <br /> &#039;&#039;&#039;attL2-R&#039;&#039;&#039;&lt;br&gt;(3&#039;TAG ctcgag ACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCcccc5&#039;)&lt;br&gt;ggggGATAAGCAATGCTTTCTTATAATGCCAACTTTGTACAAGAAAGCTGGGTctcgagCTA <br /> <br /> == &lt;br&gt;1st PCR ==<br /> <br /> 2.5 ul of 10 X Pfu PCR buffer&lt;br&gt;0.5 ul of dNTPs (10 mM)&lt;br&gt;1 ul of cDNA or gDNA (~20 ng/ml)&lt;br&gt;0.5 ul of GSS-F (10 uM)&lt;br&gt;0.5 ul of GSS-R (10 uM)&lt;br&gt;0.2 ul of Pfu (5 U/ul)&lt;br&gt;sterile water to 25 ul <br /> <br /> 95 oC for 2 min&lt;br&gt;95 oC for 30 s&lt;br&gt;55 oC for 30 s&lt;br&gt;68 oC for 6 min (1 to 1.5 min per kb insert)&lt;br&gt;15 cycles &lt;br&gt;72 oC for 5 min <br /> <br /> == 2nd PCR ==<br /> <br /> 5 ul of 10 X Pfu PCR buffer&lt;br&gt;1 ul of dNTPs (10 mM)&lt;br&gt;x ul pf 1 round PCR product &lt;br&gt;1 ul of attL1-F (10 uM)&lt;br&gt;1 ul of attL2-R (10 uM)&lt;br&gt;0.4 ul of Pfu (5 U/ul)&lt;br&gt;sterile water to 50 ul <br /> <br /> 95 oC for 2 min&lt;br&gt;95 oC for 30 s&lt;br&gt;55 oC for 30 s&lt;br&gt;68 oC for 6 min (1 to 1.5 min per kb insert)&lt;br&gt;15 cycles &lt;br&gt;72 oC for 5 min <br /> <br /> == Gel Purify ==<br /> <br /> Optional <br /> <br /> == &lt;br&gt;LR reaction ==<br /> <br /> 4 ul of LR Reaction buffer (5x)&lt;br&gt;3–8 ul of the 2nd PCR product (50–300 ng)&lt;br&gt;0.5 ul of destination vector (100–250 ng)&lt;br&gt;4 ul of Gateway LR Clonase mix&lt;br&gt;Final reaction volume: 20 ul&lt;br&gt;Incubate O/N at room temperature&lt;br&gt;&lt;br&gt; <br /> <br /> [[Category:Protocols]]</div>

Paul.xu