Hi guys. thanks for reading. I want to make transgenic line with my gene of interest using its own promoter with and without GUS reporter gene.
My first question is if iclone in my gene of interest together with its endogenous promoter with and without stop codon, can I consider the construct with codon stop as endogenous promoter lines without GUS tag? As I will expect no GUS staining from the construct with its native stop codon right after CDS.
My second question is if I run RT-PCR will I still able to detect GUS transgene in transgenic with stop codon? Im using pBI101 vector that have a MCS before GUS reporter gene. Thank you very much!
Question 1 & 2 revolve around the same issue. As you're working in bacteria, you have to take into account that bacteria are equipped for operon structures. If there is no real transcription stop signals between your gene and the GUS mRNA information, you will have some transcripts coding for your gene and GUS. During translation the ribosome will stop at the stop codon of your gene, yet you might see some re-initiation at the GUS start codon and even some read-through. So if you really want to be free of GUS background, I would recommend transcription stops after your gene, or removing the GUS CDS as a whole.
Quick comment about RT-PCR to detect GUS. I am assuming you are talking about detecting it (or not) in plants at this point. I had very hard time trying to detect GUS transcription Arabidopsis and ultimately gave up - there is tons of homology to GUS and I could not get clean primers.
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