9 replies to this topic

[linainwerse]

Hi!
I do multiplexing Taq man assay, recently I'm having problems with 2 genes which were working perfectly fine so far (please see attached file).  I've checked mix, primers, probes, machine, template and I couldnt find solution. If anoybody had similar problem I would be grateful for any advice.

Attached Files

•  Graphs__3_.doc   66.5K   107 downloads

[Prep!]

if it is run on the same machine in the same plate using the same master mix, then those parameters can be eliminated but i think there is a dilution error in gene 1 by the look of your graphs!!!

[linainwerse]

Pradeep Iyer, on Oct 27 2009, 09:27 AM, said:

if it is run on the same machine in the same plate using the same master mix, then those parameters can be eliminated but i think there is a dilution error in gene 1 by the look of your graphs!!!

No it is not dilution error (this was the first thing I've checked), when I dilute my standard curve I have at least four templates in the same reaction, the other two are perfectly fine in the same sample. In gene 1 i have no amplification after 4th dilution - i've chcecked it several times

[Prep!]

linainwerse, on Oct 27 2009, 04:11 PM, said:

Pradeep Iyer, on Oct 27 2009, 09:27 AM, said:

if it is run on the same machine in the same plate using the same master mix, then those parameters can be eliminated but i think there is a dilution error in gene 1 by the look of your graphs!!!

No it is not dilution error (this was the first thing I've checked), when I dilute my standard curve I have at least four templates in the same reaction, the other two are perfectly fine in the same sample. In gene 1 i have no amplification after 4th dilution - i've chcecked it several times

one more thing tat i can think of is that the taqman probe for this particular gene might have lost its quantum yield and so giving almost half fluorescence!!! just a thought... were all the primers and probes purchased at the same time??

[linainwerse]

Pradeep Iyer, on Oct 27 2009, 10:47 AM, said:

linainwerse, on Oct 27 2009, 04:11 PM, said:

Pradeep Iyer, on Oct 27 2009, 09:27 AM, said:

if it is run on the same machine in the same plate using the same master mix, then those parameters can be eliminated but i think there is a dilution error in gene 1 by the look of your graphs!!!

No it is not dilution error (this was the first thing I've checked), when I dilute my standard curve I have at least four templates in the same reaction, the other two are perfectly fine in the same sample. In gene 1 i have no amplification after 4th dilution - i've chcecked it several times

one more thing tat i can think of is that the taqman probe for this particular gene might have lost its quantum yield and so giving almost half fluorescence!!! just a thought... were all the primers and probes purchased at the same time??

I've thought about it I've ordered fresh probe and it didn't solve the problem.

[Prep!]

have you repeated the entire place a second time for confirmation??? if so can you attach the amplification curve for that too??
also if you do a melt curve analysis, the data can be used to troubleshoot something. so kindly attach that too!!!

[linainwerse]

Pradeep Iyer, on Oct 27 2009, 11:30 AM, said:

have you repeated the entire place a second time for confirmation??? if so can you attach the amplification curve for that too??
also if you do a melt curve analysis, the data can be used to troubleshoot something. so kindly attach that too!!!

I've repeted this experiment several times. In the file you'll find another repeat with taq man probe I've attached another 2 genes I ran in the same sample, i've done SYBRGreen experiment as well on the same standard curve and it looks ok. I hope I've attached all information you've asked for - many thanks for help  

Attached Files

•  Gene_1.doc   232K   88 downloads

[Prep!]

linainwerse, on Oct 27 2009, 05:27 PM, said:

Pradeep Iyer, on Oct 27 2009, 11:30 AM, said:

have you repeated the entire place a second time for confirmation??? if so can you attach the amplification curve for that too??
also if you do a melt curve analysis, the data can be used to troubleshoot something. so kindly attach that too!!!

I've repeted this experiment several times. In the file you'll find another repeat with taq man probe I've attached another 2 genes I ran in the same sample, i've done SYBRGreen experiment as well on the same standard curve and it looks ok. I hope I've attached all information you've asked for - many thanks for help  

hahaha
not every one shares data so easily...  tats commendable!!!
all i can think of as of now  is if you are getting good results with SYBR green then the problem definitely has to be with something which is not common in both these chemistries!!!
BEst luck.. me thinking... do tell if sumthin good happens!!!

[Prep!]

also i can see some primer dimers or non specific binding in the gene 1 and 2 melt curves... if theya re not present in the other two genes curve, then may be you should recheck the primers one again!!! by the way if primers are same for all the genes, then this might be possible due to some sheared DNA in gene 1 and 2 and so the primer got no chance to bind as efficiently as for gene 3 and 4, if they are different (primers) may be recheck them!!!
hope i am not repeating the same things that you might have already thought of and frustrating you even further!!!  

[linainwerse]

Pradeep Iyer, on Oct 27 2009, 12:07 PM, said:

also i can see some primer dimers or non specific binding in the gene 1 and 2 melt curves... if theya re not present in the other two genes curve, then may be you should recheck the primers one again!!! by the way if primers are same for all the genes, then this might be possible due to some sheared DNA in gene 1 and 2 and so the primer got no chance to bind as efficiently as for gene 3 and 4, if they are different (primers) may be recheck them!!!
hope i am not repeating the same things that you might have already thought of and frustrating you even further!!!  

Don't worry i'm not showing any top secret data  
I use 4 sets of primers for 4 different genes, primers were validated and they are fine. I think those unspecific picks are due to template excess because they are present only in more concentrated dilutions in all genes. I'm 98% sure something is wrong with my probes  but i don't know what - as I said before they were working fine so far. i'm starting to think maybe my suplier did something wrong. Anyway - many thanks for your help