https://bridgeslab.sph.umich.edu/protocols/index.php?action=history&feed=atom&title=TOPO_CloningTOPO Cloning - Revision history2026-06-01T21:10:46ZRevision history for this page on the wikiMediaWiki 1.45.1https://bridgeslab.sph.umich.edu/protocols/index.php?title=TOPO_Cloning&diff=28&oldid=prevDavebrid: Created page with '#PCR desired insert using Pfu polymerase. Add 1 uL of Taq and mix. Incubate 10min at 72C to add A to 3’ ends. Use immediately #If PCR product is a single band, no need to pu...'2009-05-02T00:45:17Z<p>Created page with '#PCR desired insert using Pfu polymerase. Add 1 uL of Taq and mix. Incubate 10min at 72C to add A to 3’ ends. Use immediately #If PCR product is a single band, no need to pu...'</p>
<p><b>New page</b></p><div>#PCR desired insert using Pfu polymerase. Add 1 uL of Taq and mix. Incubate 10min at 72C to add A to 3’ ends. Use immediately<br />
#If PCR product is a single band, no need to purify further. If several bands, gel purify.<br />
#Combine 4 uL PCR product, 1 uL Salt Solution, and 1 uL TOPO vector. Mix gently<br />
#Incubate at room temperature for 5 min, place on ice.<br />
#Thaw tube of OneShot cells on ice<br />
#Add 2 uL reaction and incubate on ice 5-30 min<br />
#Heat shock 30s at 42C without shaking<br />
#Place on ice for 2 min<br />
#Add 250 uL of room temperature SOC media<br />
#Incubate at 37C for 1h<br />
#Plate 50 uL on plate and grow overnight</div>Davebrid