3:15: "you can even change the experiment type on all of the newer Life Technologies platforms". Could someone elaborate on what "newer" means? I wasn't allowed to with the StepOne Software v2.2.2. Whatever the case, If you want Ct values along with your melt curve, save yourself some trouble and add the melt curve to a "quantitation" setup type (i.e. don't run the Melt Curve type if you want Cts also). At least raw fluorescent values can be exported, so you could manually calculate Cts based on the 2nd derivative of the raw fluorescence values.
Hi im trying to run a qpcr in a step one plus equipment, it was all doing fine so when it started the stage 2 i went to my computer, 2hrs later i went back to the machine for my results and i realized that the run stoped at the 2nd cycle of stage two, i cant stop the run because even if i try the plate is not coming down and the display is not working, can someone please please help me!!!
Hello, I want to make a question regarding the analysis of Allelic discrimination plot, I performed several experiments but I´not sure about the process of register the data in the equipment and when the experiment finished it can´t show me the Allelic discrimination plot, Can I get the plot byanalizing the fluorescence data?
Hello Fenix, Thanks for your question. Can you please reach out to our technical support team at thermofisher.com/askaquestion. They would be the best team to assist. Thank you!
Hi!I did not change the original setting for me qPCR run and I read the plate as TaqMan probes when it should had been Sybr green. Can I change it afterwards? Thanks
Hello, I wanted to know if it's possible to set up data collection at different temperatures on the run. I tried to do that, but when I see the results, there's only one Ct per well and I should see 3, maybe there is a mistake on the set up of the run.
Dear sir, we are using AB 7500 Real Time PCR instrument... we want to detect a pathogen. If we need to detect whether pathogen is present or absent in a sample, which experiment we need to select... Quantitative comparative experiment, Standard curve experiment, Relative standard curve experiment or presence/absence experiment... We are started with quantitative comparative experimental method first.... we used a designed probe specific to the pathogen... If we get unknown samples, NTC, positive control and negative control, which sample should consider as the reference sample... 1st we took negative control sample (Nuclease free water) as our reference sample.... Here we got CT values for positive control around 15-16. That is ok... Unknown samples we got that number around 32.... Is it an acceptable positive sample or neglected one... I meant CT value around 32 can be consider as positive? My second question is, we got amplification for non template control (NTC) also...here CT value around again 32, 33.... Can it be happened... ? Or it might be a contamination of water? Same thing (amplification of NTC) happened in SYBRGreen chemistry experiment also.... Please help me in solving my problems... I have some doubt on selecting the experimental method of quantitative comparative...is it ok for detecting presence of a pathogen ? And another doubtful thing in my mind in selecting the reference sample... We are setting up annealing temperature to 60 celcius.... Extension step also not used for taqman probe method.... Denaturation and annealing only used.....
Dear thank you a billion for your valuable support. I'd like to ask you First, how we could be minimize the false positive, false negative and indeterminate result for covid-19 in RT-PCR(QUANTOSTIDO 7 INFLEX). Second, how to clear the blank well in RT-PCR before we are going to analysis or does it has impact on result? Third, for how long covid-19(RNA) stable and at what temperature? fourth, who we assure the calibration of RT-PCR? thank you in advance for you indispensable answer.
Thank you for your question. False positives, false negatives, and inconclusive results can be minimized by following our instructions for use if you are using our EUA or IVD/CE COVID19 kit. The Quantstudio 7 can be calibrated by the end user or by having a preventative maintenance performed by one of our engineers. The instructions on how to perform these calibrations are found in the user guide which you can find on our website. Regarding the stability and your software analysis questions, please contact our technical support team at thermofisher.com/askaquestion. Thank you.
Hi! By mistake, I turned on data collection in both stage 2 step 2(annealing) and step 3(extension)? Did it collect data in both stages and can I access Ct values collected only in stage 2 step 3? TNX!!
Please tell why some or most of the well show reading undetermined even its having template. (1) Is this a technical issue? (2) can this be corrected as I have got correct melting curves of the same?
I run multiplex qPCR and met the error: ''Error occurred during analysis due to incorrect data'' in one Chanel, other chanel were normal. How to fix it?
Hi...I have a big trouble as I running qPCR...I made mistakes by clicking "Save as template" as the machine running and the result in that file was overlapped by a empty template file...but the machine was still running(I can't click stop running in both software and machine...) It still 1.5 hours of remaining time...I have no idea if it will stop running automatically by the end... please help me...
Thank you for your question Ting Ru. If you have issues recovering your StepOne Plus data, you can contact us at thermofisher.com/askaquestion. The StepOne Plus should finish running the experiment despite you clicking “Save as template”. It may have disrupted the real time viewing on your computer, but the instrument should have continued running and finished. You can get the .eds file directly from the instrument if that’s was occurred. For additional troubleshooting, please reach us at the link above. Thank you.
Hello! I have recently run a plate and unfortunately I got my controls wrong (e.g I said they were T/T instead of T/G). The results were not very good (a lot of undetermined) and some of the samples that were previously run in others plates and we already knew their genotypes came (in this recent plate) with different genotypes (e.g we knew that sample X was G/T in previous plates and now it was T/T). Any idea of what may be happening? Thanks!
Hi..I wanted to ask...actually all my settings...everything is fine and the melt curve is also ok..bt i did a big mistake...i forgot to set the plate read after every cycle so i dont have any ct values data...but i have the melt curve data...is there any way i can get that...or shd i just set up the reaction again..
+Aj Hi, Unfortunately if the data collection was not turned on during the amplification stage then there is no way to fix this. You would have to set up a new plate to run again.
Hola en las dos últimas PCR me han salido en varios pocillos esté fallo "The software cannot identify the exponential region of the amplification plot" ¿cómo puedo solucionarlo?
Gracias por tu pregunta. Póngase en contacto con nuestro equipo de soporte técnico en thermofisher.com/askaquestion. Puede compartir el archivo con ellos y ellos pueden ayudarlo aún más con su pregunta. ¡Gracias!
This doesn't work for me. I accidentally set the reference dye on ROX while it should have been None. At 'setup', I clicked on 'None', but how do I analyse this again? At 'Analysis' I can't click on the green button 'Analyze', it's greyed out and not clickable. I use a .eds file from an .edt template. If you can help, thank you very much.
***** Thanks for the help, but no thank you, it's already fixed. The file was broken (I think it wasn't saved correctly) so I went back to lab to get another, previously saved version of the file that did work. I could analyse that other version so the problem is solved. Thanks for the offer.
i have a problem in real time pcr appearence of yellow triangle in sample plates after the end of run, i ask how avoid and if it affect my reading CTs values. In addition i have another problem that i have no melting curve at the end of pcr reation due to certain error occured in the setup of melting curve cycle.
The yellow triangles mean there is a flag on the well. There should be a ‘QC Summary’ section in your software that will go into more detail as to which flag is present and what it means. If you still have your plate, you can always go back and run the melt curve separately after the run is complete. If you have more questions, please send your run file to techsupport@thermofisher.com and we would be happy to help.
Thank you for your question Elisa. You are able to change the quencher selection after the run is done. You can open up your .eds file in the StepOne software and in the setup tab, go to the plate setup and change the quencher to what it should be. If you are using a non-fluorescent quencher (QSY) or dark quencher, you can choose None. Choosing the incorrect quencher affects the data only if you are choosing a fluorescent quencher like TAMRA and using a non-fluorescent/dark quencher. This can be rectified by changing the quencher in the software after the run. For additional technical support, please reach out to us at thermofisher.com/askaquestion. Thank you.
Hi . I used Applied Bio system PCR 7500 but during run after cycles 24 or 26 it stopped and gave below massage Fatal error occurred Hardware error (CCD fatal error 64 CCD acquisition failure) current run is aborted Kindly I ask from you to guide me what should I do?
Thank you B.Frank for your question. The software is called StepOne Software. We are currently at v2.3 and the software can be found using the link below. www.thermofisher.com/us/en/home/technical-resources/software-downloads/StepOne-and-StepOnePlus-Real-Time-PCR-System.html For additional technical support, please contact us at Thermofisher.com/askaquestion. Thank you!
Thank you for your question. May I ask what changes you made to your wells? After making a modification to your well set-up, you will have to hit analyze in order for the software to adjust for the changes you made to the well. If after hitting analyze, your wells are still blank, please contact techsupport@thermofisher.com with your file and we can assist you further with troubleshooting. Thank you! Best, Mai
Thermo Fisher Scientific at the end of the experiment everything came back! Thank u. Also I wanted To know. Does the ct value change if in the settings i put 20 ul, when I actually had 10ul?
You’re very welcome. Your CT values shouldn’t significantly change if you had 10 uL in the wells and put 20 uL in the software. The volume is taken into consideration for ramp rate purposes and since your volume was less than anticipated, your sample may have gotten to the temperature slightly faster than anticipated. However, it shouldn’t be a significant change. I hope this helps. Best, Mai
Uumm I have 2 questions And pls I'm begging you, give me a solution cause I'm driving crazy. I was using 7500 applied biosystems, but the machine broke out. So I had to transfer my protocol to a 7500 fast machine. My 1st plate run with this protocol: [95°- 3min], 95°- 15sec, 60°- 1min /40cycles My results were the same as always! But my supervisor told me to change the protocol cause that wasn't the ideal fast one for the machine. And so I did! After that my results are awful, the variants give CT above 30cycles and even HPRT 1 gives expression 27-28 I have tested everything (cDNA templates, primers, enzyme) and I haven't found any solution yet! I use SYBRgreen and my variants length is 150-240bp. My second question is about the filters in expert mode. In the fast machine we click only 1 & 4 . Now, I want to run my plate with the standard protocol, do I have to click all of them (1,2,3,4,5)??? HELP ME PLEASE!
Hi Kat. Thanks for your questions. It’s difficult to troubleshoot without knowing what your original protocol is. Could you please contact our technical support team at thermofisher.com/askaquestion and provide us both .sds/.eds raw data files and additional information regarding what specific master mix you are using? We have several different Sybr master mixes and we’d like to understand your workflow a bit more. Regarding your filters, you can select all of the filters. This tells the software to collect data for all 5 filters. If you are only using two filters and need a shorter data collection time (less than 20 seconds), you can select those necessary. Filter 1 is usually used for the FAM and SYBR dye. Filter 4 is usually used for ROX. Most customers just leave all 5 filters on and it doesn’t affect the data in any way.
OMG thank GOD I found this video. I realised I labelled FAM instead of VIC on 3 runs I did last week, so glad it's as simple as this to fix it!
GOD had nothing to do with it. Thermo Fisher Scientific produced a video, which helped you.
You just saved me 3 hours of work!
That's great Heather! Let us know if we can help with anything else.
Million Thanks yo TaqMan :)
Do you have any video about real-time PCR set up in 7900HT machine? I am driving crazy about this
3:15: "you can even change the experiment type on all of the newer Life Technologies platforms". Could someone elaborate on what "newer" means? I wasn't allowed to with the StepOne Software v2.2.2.
Whatever the case, If you want Ct values along with your melt curve, save yourself some trouble and add the melt curve to a "quantitation" setup type (i.e. don't run the Melt Curve type if you want Cts also). At least raw fluorescent values can be exported, so you could manually calculate Cts based on the 2nd derivative of the raw fluorescence values.
aaaa you saved my day! Thank you!
Hi im trying to run a qpcr in a step one plus equipment, it was all doing fine so when it started the stage 2 i went to my computer, 2hrs later i went back to the machine for my results and i realized that the run stoped at the 2nd cycle of stage two, i cant stop the run because even if i try the plate is not coming down and the display is not working, can someone please please help me!!!
Hello, I want to make a question regarding the analysis of Allelic discrimination plot, I performed several experiments but I´not sure about the process of register the data in the equipment and when the experiment finished it can´t show me the Allelic discrimination plot, Can I get the plot byanalizing the fluorescence data?
Hello Fenix, Thanks for your question. Can you please reach out to our technical support team at thermofisher.com/askaquestion. They would be the best team to assist. Thank you!
Hi!I did not change the original setting for me qPCR run and I read the plate as TaqMan probes when it should had been Sybr green. Can I change it afterwards? Thanks
Hi. I put my samples as targets and my targets as samples. Is there anything I can do? Or I have to repeat the experiment? Thanks
OMG thanks for saving my life at late night! I was about to repeating the expt.....kissssss!
Glad it was useful, thanks for watching.
Hello, thank's for your explanation,
My trouble is about Ct, I have the curves but without Ct how could I make them availbales in the results
Hello, I wanted to know if it's possible to set up data collection at different temperatures on the run. I tried to do that, but when I see the results, there's only one Ct per well and I should see 3, maybe there is a mistake on the set up of the run.
Dear sir, we are using AB 7500 Real Time PCR instrument... we want to detect a pathogen. If we need to detect whether pathogen is present or absent in a sample, which experiment we need to select... Quantitative comparative experiment, Standard curve experiment, Relative standard curve experiment or presence/absence experiment... We are started with quantitative comparative experimental method first.... we used a designed probe specific to the pathogen... If we get unknown samples, NTC, positive control and negative control, which sample should consider as the reference sample... 1st we took negative control sample (Nuclease free water) as our reference sample.... Here we got CT values for positive control around 15-16. That is ok... Unknown samples we got that number around 32.... Is it an acceptable positive sample or neglected one... I meant CT value around 32 can be consider as positive?
My second question is, we got amplification for non template control (NTC) also...here CT value around again 32, 33.... Can it be happened... ?
Or it might be a contamination of water?
Same thing (amplification of NTC) happened in SYBRGreen chemistry experiment also....
Please help me in solving my problems...
I have some doubt on selecting the experimental method of quantitative comparative...is it ok for detecting presence of a pathogen ?
And another doubtful thing in my mind in selecting the reference sample...
We are setting up annealing temperature to 60 celcius.... Extension step also not used for taqman probe method.... Denaturation and annealing only used.....
Dear thank you a billion for your valuable support. I'd like to ask you First, how we could be minimize the false positive, false negative and indeterminate result for covid-19 in RT-PCR(QUANTOSTIDO 7 INFLEX). Second, how to clear the blank well in RT-PCR before we are going to analysis or does it has impact on result? Third, for how long covid-19(RNA) stable and at what temperature? fourth, who we assure the calibration of RT-PCR? thank you in advance for you indispensable answer.
Thank you for your question.
False positives, false negatives, and inconclusive results can be minimized by following our instructions for use if you are using our EUA or IVD/CE COVID19 kit.
The Quantstudio 7 can be calibrated by the end user or by having a preventative maintenance performed by one of our engineers.
The instructions on how to perform these calibrations are found in the user guide which you can find on our website.
Regarding the stability and your software analysis questions, please contact our technical support team at thermofisher.com/askaquestion. Thank you.
Mr Taqman... If my the PC turns off in between the run anyway or disconnects then is it possible to retrieve the run data? Or re-connect?
Thanks you so much!
Just love U guys!
Hi!
By mistake, I turned on data collection in both stage 2 step 2(annealing) and step 3(extension)? Did it collect data in both stages and can I access Ct values collected only in stage 2 step 3?
TNX!!
Please tell why some or most of the well show reading undetermined even its having template. (1) Is this a technical issue? (2) can this be corrected as I have got correct melting curves of the same?
Hi Jyoti, Thank you for your question. Please contact our technical support team at thermofisher.com/askaquestion for troubleshooting. Thank you!
I run multiplex qPCR and met the error: ''Error occurred during analysis due to incorrect data'' in one Chanel, other chanel were normal. How to fix it?
Thx God! This video exist.
Is there any specific program to run for standardization of primers?
Hi...I have a big trouble as I running qPCR...I made mistakes by clicking "Save as template" as the machine running and the result in that file was overlapped by a empty template file...but the machine was still running(I can't click stop running in both software and machine...) It still 1.5 hours of remaining time...I have no idea if it will stop running automatically by the end... please help me...
Thank you for your question Ting Ru.
If you have issues recovering your StepOne Plus data, you can contact us at thermofisher.com/askaquestion.
The StepOne Plus should finish running the experiment despite you clicking “Save as template”. It may have disrupted the real time viewing on your computer, but the instrument should have continued running and finished.
You can get the .eds file directly from the instrument if that’s was occurred. For additional troubleshooting, please reach us at the link above. Thank you.
Hello! I have recently run a plate and unfortunately I got my controls wrong (e.g I said they were T/T instead of T/G). The results were not very good (a lot of undetermined) and some of the samples that were previously run in others plates and we already knew their genotypes came (in this recent plate) with different genotypes (e.g we knew that sample X was G/T in previous plates and now it was T/T). Any idea of what may be happening? Thanks!
Sorry for the delay in reply. Is this still a concern?
Hi..I wanted to ask...actually all my settings...everything is fine and the melt curve is also ok..bt i did a big mistake...i forgot to set the plate read after every cycle so i dont have any ct values data...but i have the melt curve data...is there any way i can get that...or shd i just set up the reaction again..
+Aj
Hi,
Unfortunately if the data collection was not turned on during the amplification stage then there is no way to fix this. You would have to set up a new plate to run again.
Hola en las dos últimas PCR me han salido en varios pocillos esté fallo "The software cannot identify the exponential region of the amplification plot" ¿cómo puedo solucionarlo?
Gracias por tu pregunta. Póngase en contacto con nuestro equipo de soporte técnico en thermofisher.com/askaquestion. Puede compartir el archivo con ellos y ellos pueden ayudarlo aún más con su pregunta. ¡Gracias!
This doesn't work for me. I accidentally set the reference dye on ROX while it should have been None. At 'setup', I clicked on 'None', but how do I analyse this again? At 'Analysis' I can't click on the green button 'Analyze', it's greyed out and not clickable.
I use a .eds file from an .edt template.
If you can help, thank you very much.
+Miriam Korver Hi Miriam. Can you send the .eds file to techsupport@lifetech.com so we can look into this further? What software version do you have?
***** Thanks for the help, but no thank you, it's already fixed. The file was broken (I think it wasn't saved correctly) so I went back to lab to get another, previously saved version of the file that did work. I could analyse that other version so the problem is solved. Thanks for the offer.
i have a problem in real time pcr appearence of yellow triangle in sample plates after the end of run, i ask how avoid and if it affect my reading CTs values.
In addition i have another problem that i have no melting curve at the end of pcr reation due to certain error occured in the setup of melting curve cycle.
The yellow triangles mean there is a flag on the well. There should be a ‘QC Summary’ section in your software that will go into more detail as to which flag is present and what it means. If you still have your plate, you can always go back and run the melt curve separately after the run is complete. If you have more questions, please send your run file to techsupport@thermofisher.com and we would be happy to help.
Does this work also if I set the wrong quencher?
Thank you for your question Elisa.
You are able to change the quencher selection after the run is done. You can open up your .eds file in the StepOne software and in the setup tab, go to the plate setup and change the quencher to what it should be.
If you are using a non-fluorescent quencher (QSY) or dark quencher, you can choose None. Choosing the incorrect quencher affects the data only if you are choosing a fluorescent quencher like TAMRA and using a non-fluorescent/dark quencher.
This can be rectified by changing the quencher in the software after the run. For additional technical support, please reach out to us at thermofisher.com/askaquestion.
Thank you.
I get an error with java, could not load virtual machine. Any help would be appreciated.
Thanks for the question, do you have any other information you can provide so that we can better assist you?
Thermo Fisher Scientific this was a fresh installation and it returned an error “ java virtual machine could not load”
Thermo Fisher Scientific it’s a windows 7 OS
Hi . I used Applied Bio system PCR 7500 but during run after cycles 24 or 26 it stopped and gave below massage Fatal error occurred Hardware error (CCD fatal error 64 CCD acquisition failure) current run is aborted Kindly I ask from you to guide me what should I do?
Thanks for your question, we'll get right back to you.
What is the name of the software you are using?
Thank you B.Frank for your question. The software is called StepOne Software. We are currently at v2.3 and the software can be found using the link below.
www.thermofisher.com/us/en/home/technical-resources/software-downloads/StepOne-and-StepOnePlus-Real-Time-PCR-System.html
For additional technical support, please contact us at Thermofisher.com/askaquestion. Thank you!
Changed the wells during the pcr experiment and everything dissapeared. What can I do?!
Thank you for your question. May I ask what changes you made to your wells? After making a modification to your well set-up, you will have to hit analyze in order for the software to adjust for the changes you made to the well. If after hitting analyze, your wells are still blank, please contact techsupport@thermofisher.com with your file and we can assist you further with troubleshooting. Thank you!
Best,
Mai
Thermo Fisher Scientific at the end of the experiment everything came back! Thank u. Also I wanted To know. Does the ct value change if in the settings i put 20 ul, when I actually had 10ul?
You’re very welcome. Your CT values shouldn’t significantly change if you had 10 uL in the wells and put 20 uL in the software. The volume is taken into consideration for ramp rate purposes and since your volume was less than anticipated, your sample may have gotten to the temperature slightly faster than anticipated. However, it shouldn’t be a significant change. I hope this helps.
Best,
Mai
Thermo Fisher Scientific thank you!!!
Thanks dear
Uumm
I have 2 questions
And pls I'm begging you, give me a solution cause I'm driving crazy.
I was using 7500 applied biosystems, but the machine broke out.
So I had to transfer my protocol to a 7500 fast machine.
My 1st plate run with this protocol: [95°- 3min], 95°- 15sec, 60°- 1min /40cycles
My results were the same as always!
But my supervisor told me to change the protocol cause that wasn't the ideal fast one for the machine.
And so I did! After that my results are awful, the variants give CT above 30cycles and even HPRT 1 gives expression 27-28
I have tested everything (cDNA templates, primers, enzyme) and I haven't found any solution yet!
I use SYBRgreen and my variants length is 150-240bp.
My second question is about the filters in expert mode. In the fast machine we click only 1 & 4 . Now, I want to run my plate with the standard protocol, do I have to click all of them (1,2,3,4,5)???
HELP ME PLEASE!
Hi Kat. Thanks for your questions. It’s difficult to troubleshoot without knowing what your original protocol is.
Could you please contact our technical support team at thermofisher.com/askaquestion and provide us both .sds/.eds raw data files and additional information regarding what specific master mix you are using?
We have several different Sybr master mixes and we’d like to understand your workflow a bit more.
Regarding your filters, you can select all of the filters. This tells the software to collect data for all 5 filters. If you are only using two filters and need a shorter data collection time (less than 20 seconds), you can select those necessary. Filter 1 is usually used for the FAM and SYBR dye. Filter 4 is usually used for ROX.
Most customers just leave all 5 filters on and it doesn’t affect the data in any way.
Thank you!!