the only pointers here are:
1. I use DTT free setup (because my constructs need no DTT) also I have glutathione in my master mix. will that affect the robust eGFP?
what can be the possible reasons. the rest of the reactions are still working fine. any other hits other than DNA (since I have tried 3 different eGFP constructs but no luck)?
This question makes no sense. No one can tell what it is you are actually trying to do. Cell free reaction for what?
cell free reaction for my protein of interest but the positive control is not showing up positive result. how does it matter what for I m using cell free. Question is straight fwd if positive control doesnt work
No the question is not very specific at all. Are you doing an in vitro transcription/translation reaction to make your proteins? Use the correct terminology. What is your assay for the proteins you are making, western blots maybe? Lastly, it is best not to insult people who are trying to help you.
well I am performing in vitro transcription-translation. (continous batch).
My protein of interest has several disulfides. hince I use cell free setup conditions with no DTT, in the presence of GSH buffers. with every reaction I put eGFP as positive control. but somehow in these set of reactions (which I m repeating) the eGFP is not getting expressed (which was working fine). However, the test is expressing. having no singnal in positive control is quite misleading and disappointing too. however, I am checking back the sequence of the eGFP constructs I have. also I am now repeating with 2 separate master mixes (for eGFP and for test). but always the eGFP had shown expression even in the absence of DTT n presence of GSH buffers. just because I was not being able to figure out if it is a human error during setting up the reaction or its some technical flaw in the plan or if anyone has experienced it so far as well. my sequencing results would be a better answer though.
Lastly, I never meant to insult. My sincere apologies to you Gregory.
Thanks
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