Problems with golden gate/voytas
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megb...@gmail.com
Jul 9, 2013, 6:12:41 PM7/9/13
to taleff...@googlegroups.com
Hello,
I am trying to assemble a TALEN using the Golden Gate kit from the Voytas lab. I have attempted the first reaction. This resulted in a vast majority of blue colonies for both A and B plasmids. What little white colonies were there were negative in the colony PCR and had strange bands in the double digest. I have added ATP with older buffer, I have used new buffer. I am including the plasmid free nuclease step. All of the enzymes are new. We digested the RVD plasmids with BsaI to check that BsaI is actually cutting. We do get a band at 3kb, which we expect, but also get bands at 4kb and 2.5kb; I don't know why. I suppose it's possible that this is due to backbone rearrangements. Is this why I get no positive colonies in the first reaction or is there something else? Please help. Thank you.
Megan
I am trying to assemble a TALEN using the Golden Gate kit from the Voytas lab. I have attempted the first reaction. This resulted in a vast majority of blue colonies for both A and B plasmids. What little white colonies were there were negative in the colony PCR and had strange bands in the double digest. I have added ATP with older buffer, I have used new buffer. I am including the plasmid free nuclease step. All of the enzymes are new. We digested the RVD plasmids with BsaI to check that BsaI is actually cutting. We do get a band at 3kb, which we expect, but also get bands at 4kb and 2.5kb; I don't know why. I suppose it's possible that this is due to backbone rearrangements. Is this why I get no positive colonies in the first reaction or is there something else? Please help. Thank you.
Megan
woos...@umn.edu
Jul 5, 2016, 4:38:21 PM7/5/16
to TAL Effectors Discussion Forum
This happened to me as well. If there are any Golden Gate experts who could help, this would be very much appreciated.
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