GEX Second Strand Buffer

[jgibbons1]

This is the second strand buffer provided by the Illumina kit.

Does anyone have a recipe for it?

Many thanks,

John

[plantseq]

Neb

Hey John,
the Illumina second strand synthesis is not so different from the NEB one. There the buffer is composed of 10mM Tris-HCL (pH 7.5), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol.
However, I would guess that Illumina also puts some E. coli DNA Ligase already in the buffer. If you want to use that buffer I would add 6.000 units/ml DNA Polymerase I, 5.000 units/ml RNase H and 25.000 units/ml E.coli DNA ligase to the mix.
Good luck,
Ben

[jgibbons1]

Awesome! Thanks Ben...

I've been using a similar recipe with pretty good:

500mM Tris-HCL (pH 7.8)
50mM MgCl2
10mM DTT

I'd like to try yours out though. I'll let you know if there are any major differences.

Thanks,

John

[plantseq]

Hey John,
the buffer described previously is only the solution the enzymes are kept in. Here is the reaction buffer you put them in together with your single strand cDNA. Sorry about that,
Ben

1X NEBNext Second Strand Synthesis Reaction Buffer:
20 mM Tris-HCl
12 mM (NH4)2SO4
5 mM MgCl2
0.16 mM β-NAD
0.19 mM dNTPs each
pH 7.4 @ 25°C

[BSP13]

Hey all,

sorry for digging this thread but I have a related question:
I want to perform a second strand synthesis of my cDNA. Originally, I planned to use the Invitrogen Superscript System but it is too expensive for my project budget. So I ordered reagents separately and wanted to use the NEB Buffer. The only difference I could spot is that NEB doesnt use KCl (final concentration in Superscript System is 90mM). NEB states that their System is suitable for 10-100ng of template. Invitrogen states that templates up to several ug are possible. So my questions are: what is the role of KCl in the Buffer and is there any connection between it's application and the template restriction?

Thank you for any help here!