Hi,
I want to multiplex some reaction with dual label probes.
I check the probes on FAM, and HEX channel. When I change the dye for ROX channel I don't see any fluorescent. I ordered the probes from two different companies, and both doesn't work.
I want to mix 3 probes in my reaction for FAM, HEX and ROX channel
Hi Elzbieta Lenartowicz which instrument did you use? Sometimes instruments use ROX like reference dye...
Try to change ROX. For example...Cy5 or Cy5.5.
May be I do not understand you well...but are you sure in your ROX-probe quality. I mean design...It could be a problem if no ).
Please give me more information
I would suspect instrument optics failure around 602nm range if filters configuration on your RT PCR machine allow to detect all 3 dyes. Do you use the same primers limiting strategy between all multiplex reactions and dyes?
Natallia Mikhalkevich
Andrei S. Babenka
thanks for reply
I use CFX96 and the dye is Texas Red. Before I used the same probe labeled with FAM or HEX and it went well. When I chenged dye, I don't see the fluorescent.
For now I stoped with the single reaction, I didn't multiplex, because I don't see the curve :(
I disolved the probes in water instead of TE - do you think it could be problem for that dye?
I found in some troubleshooting, that I can check the quality of the probe using DNase I, I will prepare this reaction tomorrow.
Texas Red hydrolysis can be a problem.
From JAMES W. GODING, in Monoclonal Antibodies (Third Edition), 1996 :
"Texas Red’ is a sulfonyl chloride derivative of sulforhodamine 101 (Haugland, 1992; Titus et al., 1982; Table 12.1)......Because of its extreme susceptibility to hydrolysis, Texas red is sold in small sealed tubes, and it is recommended that each tube should be discarded after opening and not resealed".
Greg T. Hermanson, in Bioconjugate Techniques (Third Edition), 2013
"2.10 Texas Red Hydrazide
Texas Red hydrazide is a derivative of Texas Red sulfonyl chloride made by reaction with hydrazine (Invitrogen). The result is a sulfonyl hydrazine group on the No. 5 carbon position of the lower ring structure of sulforhodamine 101. ...Texas Red hydrazide is soluble in DMF and may be dissolved as a concentrated stock solution in this solvent prior to the addition of a small aliquot to an aqueous reaction medium. The solid and all solutions made from it must be protected from light to avoid photo-decomposition. Prepare the stock solution fresh immediately before use."
Kamalika Mukherjee, Susan L. Bane, in Methods in Cell Biology, 2013
''1.2.4.2 Preparation of fluorophore stock
Texas Red hydrazide: Dissolve Texas Red hydrazide in DMSO to a final concentration of 5 mM or higher. Determine the concentration spectrophotometrically using an extinction coefficient of 109,000 M− 1 cm− 1 at 588 nm. The stock solution can be stored at − 20 °C. Vortex well before use. Keep in the dark when not in use.''
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