https://eln.excellgen.com/index.php?action=history&feed=atom&title=MS_5_5_2017MS 5 5 2017 - Revision history2026-10-18T06:20:40ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=MS_5_5_2017&diff=15869&oldid=prevCrm at 19:44, 19 May 20172017-05-19T19:44:48Z<p></p>
<p><b>New page</b></p><div>Subcloning:<br />
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1.prepare plasmid<br />
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2. 15ul per tube<br />
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3.10X Fast Digestion Buffer 1.5ul/tube (4oC)<br />
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4.Nor I and KPN I restricted digestion, 0.3ul/tube<br />
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5.spin to get rid of bubble.<br />
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6.5ul mix/tube. If the concentration is high, add 5ul H2O and 5ul plasmid. If the concentration is low, add 10 ul plasmid. Make sure 15ul/tube.<br />
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7.spin to get rid of bubble.<br />
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8.37oC incubation, 30~60 min.<br />
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9.add 5ul GRGreen loading Buffer.<br />
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10.Use 5ul Fluorescent DNA Ladder II, EG-258 as ladder.<br />
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11.Run agarouse gel, 150V<br />
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12.cut down the strip of interest, add 1ml QG buffer, disolve in 90oC water.<br />
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13.transfer liquid into mini column, spin at 13,200 rpm for 1 min.<br />
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14.wash with 350ul PE buffer, twice. then spin again.<br />
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15.wash with 20ul ddH2O.<br />
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16.add 2.5ul 10X T4 LB<br />
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17.add T4 ligase 1ul.<br />
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18.incubate in RT, 1h<br />
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19.transfer into 100ul NEB Turbo, incubate on ice for 15min.<br />
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20.Heat shock at 50oC, 2min.<br />
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21.add SOC 500ul, culture at 37oC for 30min.<br />
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22.spin down cell, drop off supernatant, resuspend and transfer to LB/Amp solid media.<br />
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[[Category:My ELN]]</div>Crm