efficiency problem - HKG's lower than target genes - (Dec/06/2005 )

I can only tell you what works for me, and I am sure not an expert! but I hope I have helped. I started 'from scratch' too and it was a frustrating PIA for a long time

from the time I first went through the technology...attended a couple lectures, read user bulletin #2 and combed the net.... asked a few techs in other labs where to start...wrapped my brain around all the technology and picky crap I would have to do to set it up properly...tackled primer design...isolated my first RNA...actually attempted a plate, was probably about 2 to 3 months (I was doing other things, but probably 1/2 of my time was spent just getting started. I was very intimidated, and my first RNA prep was not so wonderful (I learned all about formaldehyde gels too )

from then, it was about a month or 6 weeks until I had the first 4 genes and the one housekeeper that worked consistently from plate to plate and gave good reproducible results. I then spent quite a bit of time over several months fine-tuning my data analysis process; the first month or so I did it by hand and WOW that sucked...

we are also a po' little lab and some of the time was spent calculating costs and coming up with a total protocol that would save money and still not waste too much time. but overall, for me, the learning curve was long before I felt like I could trust all my data and I pretty much felt like a brain-dead loser during a lot of the initial weeks...the technology seems like it should be so easy, but there's so much to it

anyways, I can't answer for anyone else, but I don't think a couple months is unreasonable if you are taking care to do things right. it's better than having to go back later, and there are a LOT of little places to make mistakes

let me know how your run works out, if the cDNA is OK?

A

Soluene, don't be too surprised that I wrote a novel for my reply...I'm pissed off at my boyfriend and trying to kill a bunch of extra time in the lab this weekend

lol

You being pissed at your boyfriend is to my advantage!!

I'm also using work to take my mind off personal issues this weekend, and I've been spending the day in my PJ's at these boards and other websites

Thanks again, though, your posts make me think I'm on the right track.