Hi everybody,

I have tested a kit for cell free protein expression (Next generation cell free protein expression kit, wheat germ CFPS 700) from Merck and I didn't get the expected yield for protein production.

In the procedure of this kit you have to prepare DNA template by a game of several PCR, then in vitro transcription is realized from PCR template, and finally cell free translation using wheat germ extract.

All is good until transcription (agarose gel checking)

But after that the protocol is a mRNA purification using amonium acetate salt and ethanol.

I think these step is the problem because I loose a lot of mRNA.

Can somebody tell me if this step is necessary or if I can try to translate without mRNA purification? Or else, is there another methode for mRNA purification, that preserve its quality for the following transcription (the kit exclude phenol, trizol or ammonium sulfate purification that rendered mRNA unsuitable for translation)?