RT-PCR Help - Protocol provided (Sep/01/2009 )

For now, all i want is signal, but im apparently not getting any!
I'm Using GFP +Arabinose RNA- (DNased(and confirmed DNasing through PCR) promega DNase)

RT Protocol using Biorads iscript Reverse transcriptase and SYBR Green

Target Reaction
2 ul RNA
1.5 ul EcoR1 Primer
1.5 ul Xma Primer
25 SYBR Green
20 H2O
1 iScript

Control Test to see if contaminating DNA persists and is giving false signal in target
2 ul RNA
1.5 ul EcoR1 Primer
1.5 ul Xma Primer
25 SYBR Green
21 H2O
0 iScript

RT-PCR Parameters
10' @ 50*C (currently trying 40*C also)
5' @ 95*C

30" @ 95*C
30" @ 61*C
30" @ 72*C

I've tried increasing primers, template, but still no signal. Does anyone have any tips on what to change?

-dreww-

no one has any tips?
some background info
-the primers cut at the restriction sites
-the RT reaction is called single step so all i have to add is the reverse transcriptase(iscript), primers, template, and the SYBR Green(w/ polymerase), and fill to volume with water.
-the RNA if from the Green Fluorescent Protein

should i also stain the gel with EB? SYBR already has dye in it.
i PCRed dna using the SYBR and the same gene but DNA and it seems to work fine. so its the reverse transcription that isnt working!!!!!!

-dreww-

Hi dreww,

It seems that you are doing real time PCR.

I suggest that you check your tube compability with your PCR machine.
PCR tubes for normal PCR is not compatible for Real-time machine, each real-time machine got their own tubes to use.

-adrian kohsf-

dreww on Sep 3 2009, 02:25 AM said:

If you think the RT isnt working, change to a 2 step protocol.
Also, check your RNA quality.

I've never done one-step RT-PCR because I've always wanted to amplify more than one product from my RNA. But I'd think a 2 step protocol will most probably sort your problem.

Hope this helps.

-almost a doctor-

what is the reasoning behind 2 step vs 1 step?

-dreww-