4 replies to this topic

[dreww]

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!!!!!!

[Adrian K]

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.

[almost a doctor]

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

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!!!!!!

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.

[dreww]

what is the reasoning behind 2 step vs 1 step?