Positive sample NOT working well in SyGreen PCR- Inibition? - (Jul/01/2008 )
Hello,
I am developing a SyBrGreen PCR for a pathogen detection, using a new primers that have designed for this objective. My results are good for my samples (one from bacteria DNA isolated from a plate, an the other one from DNA tissue isolation) but not for a sampling that I am using as a positive control which it was very positive in histology and with an other conventional PCR. I have got also the target sequence of this sample and it is identical to the two other samples working good, then doesn't look like a primers problem. Someone has some idea of what can be the problem?!! To much pathogen DNA concentration maybe?! Or to much other DNA or interferences (is tissue DNA isolation)...??- Well I have repet the same SyBrGreen PCR with several 10 fold dilutions of positive control and 1/10 and 1/100 dilutions got a Ct of 30.24 and 35.00 respectevely. However I got undeterminated Ct values for the indiluted sample and for the 1/1000 dilution. The dissocition curve and the amplification plots are nice for this two low working concentrations.
For me it looks like it is somethink inibiting this QRT-PCR with this particular sample. But what? What I can do to improbe it?
Thanks very much in advance,
Maria
I'd guess you are using way too much DNA. Try a 100x and 1000x dilution of your positive sample.
Thanks very much for your advice, in fact I have done already that and I got better results (amplification plot and dissociaton plots were nice) but Ct were not satisfactory having in acount that this sample is very positive, I mean that has a lot of the target pathogen...:
-10X Ct around 30
-100X Ct was around 35
-1000X Not Ct
Some other suggestion?
Thanks again,
Maria
When optimizing my PCR, I have found that template copy #, primer concentration, and to some extent anneal temperature will all have an effect on the c(T). Have you optimized these factors (apparently you are working on the template copy#)? What is the purity of your template? How long is your amplicon, and what is the length of your Amplification times?
First of all thanks very much for trying to help me!
I am now trying to optimized the annealing temperature (I was thinking to try 58, 59, 60, 61 and 62 degrees), to see if I can get better ct results with this particular sample. And later on the optimization of primers concentration...
About my template, what you mean which is the purity of my template? How I can know this?
My amplicon is 60 bp and I the program is:
37ºC- 10 min
95ºC- I think 5 min but I don´t remember for sure I tell you tomorrow...
40 cicles of:
95ºC-0:15 I have also to check better those ones tomorrow
59ºC-1 min
And the addiccional dissociation stage.
I was also asking myself if the UDG can have also an effect in my problem and I have may be to optimizate also the quantity of UDG and SyBrGreen ready mastermix I use.....
What do you think??
Thanks,
Maria
Are you running one step RT-PCR using sybr green? why you run at 37C for 10 min?
Hi!
No, I am working with DNA as a template, not RNA. The 10 min at 37C step if you the rigth working of the UNG which is recomended to use to avoid contamination.
About the program I am using I checked better and it is:
37ºC- 10 min
95ºC- 10 min
40 cicles of:
95ºC-0:15
59ºC-1 min
And the addiccional dissociation stage (95C 0:15 min, 60C 1 min and 95C 0:15 min)
Thanks to all!
Maria
I have no idea what "additional dissociation stage" is about, but the program should look something like this:
37 for 10 min
95 for 2 min
<loop 40 times>
95 for 15 sec
55 for 15 sec
72 for 15 sec (lengthen for a longer product)
<end of loop>
72 for 5 min
It is a testament to the robustness of PCR if you got any product with the previous program.
37 for 10 min
95 for 2 min
<loop 40 times>
95 for 15 sec
55 for 15 sec
72 for 15 sec (lengthen for a longer product)
<end of loop>
72 for 5 min
It is a testament to the robustness of PCR if you got any product with the previous program.
Do you use this program for SyBrGreen Real time PCR? In fact we have an Applied Biosystemps 7300/7500 Real Time PCR system and we use the protocol that is recomended for the manufacturer. Many people use this program getting nice results. In fact I am getting also nice results in almost all the samples but I have a problem with this particular sample and I try to know which can be the problem... It looks like something inibiting the reaction in this particular sample giving less good results than the expected for a high positive sample.
May be I will try you program, just to see what happen.
All the best,
Maria
37 for 10 min
95 for 2 min
<loop 40 times>
95 for 15 sec
55 for 15 sec
72 for 15 sec (lengthen for a longer product)
<end of loop>
72 for 5 min
It is a testament to the robustness of PCR if you got any product with the previous program.
Do you use this program for SyBrGreen Real time PCR? In fact we have an Applied Biosystemps 7300/7500 Real Time PCR system and we use the protocol that is recomended for the manufacturer. Many people use this program getting nice results. In fact I am getting also nice results in almost all the samples but I have a problem with this particular sample and I try to know which can be the problem... It looks like something inibiting the reaction in this particular sample giving less good results than the expected for a high positive sample.
May be I will try you program, just to see what happen.
All the best,
Maria
I was thinking in your programm... which system/machine and ready mix are you using?
Thanks very much,
Maria