Trof

It depends on why your success rate is what it is. i suppose there are two reasons why an experiment goes sour. (i) error in execution and (ii) error in design.any experiments require careful planning. having stated the obvious, i do all my thinking/planning before the experiments, write down exact game of plan before you actually execute. once i am doing the experiments, i just follow my protocol exactly. i always tell my students to think about the experiments. what i mean is that i picture myself doing each steps of the protocol the night before. however, i have to admit that sometimes i found myself doing something terribly wrong. then i guess you have to think on your feet to figure out the problem. to develop skills to think on your feet, it is important to know why you are doing each steps. when you know why you are doing what you are doing, it is easier to fix your mistakes. if it is an experimental flaw, then you are doomed to begin with. it doesnt matter how well you have executed your experiments, if you are missing controls, or going after leads that are false, what is the point?

There is a learning curve to this, and it is a steep one. It's sometimes too pricy to learn by trial and error, but your perseverance will one day translate to good science.

i dont usually believe in god, but if you are up there, please save me superman! --homer simpson

my best,

jeff kwak

The secret to finding your reverse primer in your sequence:  http://www.bioinform...s/rev_comp.html

You should normalize your RNA according to grams (weight), not according to the volume.

We usually take 100 ng - 1 ug of RNA before synthesizing cDNA. Then we normally take 1- 2 ul of the cDNA reaction for our qPCR reaction, without measuring cDNA concentration. It works fine for us. It is difficult to measure cDNA concentration since it has RT enzyme, dNTP and buffer contamination. It's up to you however to measure it or not. you can also purify your cDNA before qPCR, but you will lose a lot of cDNA. If you want to measure cDNA concentration remember to adjust your spectrophotometer (for example Nnopdrop) to ssDNA.

If the template RNA is total RNA from cells make sure you use DNase to remove genomic DNA, because although you are using RNA extraction kits, it might still contain genomic DNA that might cause prroblem during qPCR as your primers might bind to gDNA and give wrong signal.

Home work?

BTW, welcome to Bioforum and the wikipedia black out is over.

As tea-test has been so great in giving us so much useful information, I thought I would also add a link that was about RNA cleanup but on the Microarray forum and given by Huni. It's not quite about qPCR but related as it is about prep of RNA for applications like qPCR and I (as a newbie to qPCR) thought was very good.

huni, on 04 January 2010 - 08:53 PM, said:

Here is the article:
 Krebs et al cleanup of TRIzol from RNA.pdf   217.88K   513 downloads

I have been using a new kit from Zymo called the [url="[url]http://www.alkaliscientific.com/en/zyppy-plasmid-miniprep/515-zyppy-plasmid-miniprep-kit.html"]Zyppy[/url] Miniprep kit[/url]. Its way faster than standard minprep kits because it bypasses the conventional pelleting and resuspension steps. It has really high yields too.. way better than Qiagen. I buy it through Alkali Scientific because they have a good price on it and free shipping.