RT-PCR primers for E. coli LacZ gene - (Dec/20/2005 )
I need your help.am a research scholar working on E coli. I had designed primers for amplification of lacZ gene from E coli and they worked now I have to do a RTPCR using total RNA isolated from E coli , with RTPCR primers specific for lacZ gene how do I go about it . I cannot use the PCR primers for RT. Please tell me the correct way of doing it.
Thanking everyone who takes time out to help me.
please reply soooooooooooon
Why can't you use the pcr primers for your RT? And why do you actually want to do RT? E. coli has no introns or any of that, so PCR on DNA is good enough, not?
I WANT TO MAKE cDNA then again do a PCR for amplifying lacZ gene and then I want to use the PCR product as a probe i...e I want to spot on glass slide to make an array
Use random hexamer or random decamer primers for reverse transcription and then amplify with the gene specific primers for PCR.
Saturate your RNA with DNase I after the extraction to prevent genomic contamination from interfering with your results.
I still have no idea why you need to do it this way for a microarray, and I work with microarrays.