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Old 05-12-2015, 03:12 AM   #15
kobeho24
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Location: HKUST, Hong Kong

Join Date: Apr 2015
Posts: 32
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Quote:
Originally Posted by Simone78 View Post
Have you tried to do the assembly in solution as we describe in the paper? Just elute the tn5 from the column and add the oligos in equimolar conc with the Tn5 --> incubate 37 degrees --> ready to use. If that doesn´t work then you might have an issue with the Tn5.
Best,
Simone
Actually, I did assemble the transpososome in solution @ 30C/RT for 1hr. Cuz I used to assemble a commercial unaasembled tn5 Tnp in this way. BTW, I was not only following the published paper but also the in-house protocol generously provided from your lab. So far, I might have to wait the arrival of the suggested strain, try one more time and see if it works. And, I was wondering that the incubation temperature after IPTG induction described on the Addgene webpage is 30C while your protocol shows that it should be 23C. As the temperature is crucial for polypeptide folding a correct way. I wanna make sure just in case. Thanks a lot!!!

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