Dear All
I am going to set up Gibson Assembly reactions. The reaction takes place in isothermal buffer which requires 100mM NAD and I have not been able to find this concentration of NAD. I would appreciate if those of you who are already doing this experiment, would let me know where to get it from. NEB sells NAD but it is a 200uL vial with just 50mM concentration.
I would appreciate your help.
Thank you
Ikram
Personnally, I use the commercial Gibson assembly master mix from NEB. No need to add anything except your DNA and water.
Are you trying to set up a home-made Gibson assembly reaction ?
If you want to save money but still use the commercial kit to avoid possibly complicated troubleshooting, know that you can greatly reduce the reaction volume that is stated in the commercial datasheet. NEB indicates reaction volumes of 20uL (thus 10uL of 2X gibson assembly master mix per reaction) and subsequent transformation of 2uL of this reaction into competent bacteria, but I scale it down to as little as 2-3uL total reaction volume (1.5uL of 2X mix) and transform the whole reaction into bacteria. This way, I can make at least 5 times more reactions with one kit.
@ Benoit,
I am interested in this topic too. I am curious: Gibson Assembly is used to fuse > or = 2 DNA fragments together. So, if assembled, it supposes to be 'linear' form of a DNA fragment. And, I am confused by your saying of 'then transform them into bacteria'. Thanks.
Thank you Yuan-Yeu for your remark. I said that because I usually assemble a linearized plasmid along with one or more overlapping fragments. In such reactions you obtain some circularized plasmid molecules containing your assembled fragments, and these are the molecules that will be transformed into bacteria. But in the case where you want to assemble fragments without reconstituting a circularized plasmid, then transformation is out of topic.
Dear Benoit
Thank you for your comment. However, I am trying the home made reaction, Thanks anyways!
Dear Ikram,
for NAD, you could order it as a powder to obtain a concentrated liquid stock ? Sigma sells it and it's quite cheap (N7004 catalog number). I don't know what is the purity you need though.
@ Benoit,
That is a great approach and makes perfect sense! Thank you for your explanation!!
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