A couple of months ago, I ran out of Invitrogen’s 5x Second Strand Buffer. I really wanted to complete cDNA synthesis on that day. Also, that buffer is unbelievably expensive for a buffer ($133/0.5 mL!). So, I looked for a recipe in my bible (Current Protocol in Molecular Biology) and yes, I found it. Here it is:
- 94 mM Tris-HCl pH 7.0
- 453 mM KCl
- 33 mM MgCl2
- 50 mM (NH4)2SO4
I made from 1 M stocks (for each component) and passed through a 0.22 µm disc filter. And, then I boiled at 100C for 20 min to inactivate DNases. Worked well!
The Ikegami laboratory @ Cincinnati Children's 