Hello!
I am wondering whether if I use FastDigest Green in a T4 DNA ligation reaction, will it work. On Fischer's website it says that the buffer is compatible for downstream applications, but I am not sure if that applies to normal FastDigest or both FastDigest Green.
Essentially, I am wanting to take cut DNA in FastDigest Green buffer, and ligate them in a separate T4 reaction with the T4 buffer.
Thanks
Adam Radek Martinez Hi,
Yes it absolutely works for the ligation, i have used this. and it worked in my case. Go ahead with this but also keep other options open.
Good luck
Somehow the question doesn't seem to be framed well.
As long as you are using T4 ligation buffer in the ligation reaction, it will work (ATP being essential but unstable component of it).
FastDigest Green is the same as FastDigest buffer with dye in it, it just avoids mixing loading dye while loading on the gel.
Ya, in the website it says "DNA/RNA modifying enzymes, such as ligases, phosphatases, kinases and mesophilic DNA polymerases also have 100% activity in FastDigest Green Buffer" which is again confusing. But don't use FastDigest-Green buffer for the ligation reaction (it has no ATP).
I hope you are purifying the digested band from gel > eluting in TE-buffer and then going ahead with Ligation.
Good Luck :)
Oh to clarify. I am not gel purifying afterwords. I want to use FastDigest Green to digest my products and then ligate directly.
I am worried that the dye may interfere with the ligation or transformation down the line. Is this the case?
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