5 replies to this topic

[Ozeir Kazemi]

hi all
I've been doing Real-Time PCR  for a month. i'm trying to trace the Lactate Dehydrogenase virus through ORF-7(Open Reading Frame 7) of the virus genome. I have recently designed a set of primer to amplify a fragment gene present(84 bp) in an cDNA template. I did classical PCR after reverse transcription reaction resulting in sharp bands in the well of 1% agarose gel. I used the same samples (which had been led to the sharp bands in PCR) in Real-Time PCR for relative quantification but I got very wired results. i wander how can I optimize my Real-Time PCR reaction based on what I did in Normal PCR? what is the best cDNA and Primer concentration?

any help would be appreciated
 1.pdf   52.73K   129 downloads
my  protocol in normal PCR

5x Buffer 10µl                
Template 2µl( 4 differnet concentration 500 ng, 200 ng, 100 ng and 50 ng of cDNA) 50 ng had most less primer dimer
P F(20µl) 1µl
P R(20µl) 1µl
Tag Pol 0.8 µl
H2O  36.2 µl
Cycles PCR
(35 cycles) :
   95 °C– 1 min
   95 °C– 15 sec
   55 °C– 15 sec
   72 °C– 10 sec
   72 °C– 10 min
4 °C –
MY Real-Time pcr Protocol

Buffer 10µL
RNAase free Water 4µL
sample  

2µL

primer F 2µL
Primer R 2µL



MeteorTaq activation 5 min. 95 °C
40 Cycles 15 sec. 95 °C
1 min. 60 °C


here some of my result in Real-Time PCR
 1.pdf   52.73K   129 downloads
 2.pdf   52.71K   112 downloads

[almost a doctor]

Have you run the products of your Real-Time PCR on a gel?    Do they show anything?    

The conditions of each reaction (normal vs real-time) are quite different, particularly the primers concentration.  Assuming the stock is at 20uM (as you wrote 20ul and I'm not sure what that means).  

For the Standard PCR you are using 392pM  while for the real time you are putting 2uM of primer.  
Your template concentration is also completely different (again here I'm assuming as you haven't actually said what volume you've added for the real time reaction) as you are using 2ul of template regardless of the final volume.

Finally, the cycling conditions are different too, I don't really understand why you'd expect to completely different reactions to work the same (regardless of the sample and primers being the same).

Another minor detail, is the volumes of your standard PCR add up to 51ul, so your buffer concentration is not 1x but I'm sure this is not a major problem.  

In my opinion it'll be the primer concentration as you seem to be using way too much in your real time PCR.

[Ozeir Kazemi]

hi, thanks for your reply
my PCR had set up for 35 cycles.

this is the protocol that i used for rt-pcr
2x reaction buffer 10µL1x
nuclease free Water 4µL
sample 2µL
primer F 2µL
Primer R 2µL

for cdna concentration of real-time pcr reaction i used concentration ranging from 50ng-200ng  with different primer concentration 1/20, 1/15, 1/10 from 100 µM (micro-molar) stock solution. i'm using nuclease free water not RNAse free water,( does this make any problem?) and i got ct value32.98, 35.48, 33.92 for my three negative controls ( using nuclease free water+ control buffer 1x+primer) and got ct value 9.5( 50 ng cDNA with 50 µM primer) and 9.7(50 ng cDNA with 50 µM primer) which are so strange and the rest of the samples including also my housekeeping gene( GAPDH) didn't reach the threshold line. my final volume is 20 µl

[Ozeir Kazemi]

for cdna concentration of real-time pcr reaction i used concentration ranging from 50ng-200ng with different primer concentration 1/20, 1/15, 1/10 from 100 µM (micro-molar) stock solution. i'm using nuclease free water not RNAse free water,( does this make any problem?) and i got ct value32.98, 35.48, 33.92 for my three negative controls ( using nuclease free water+ control buffer 1x+primer) and got ct value 9.5( 50 ng cDNA with 50 µM primer) and 9.7(50 ng cDNA with 50 µM primer) which are so strange and the rest of the samples including also my housekeeping gene( GAPDH) didn't reach the threshold line. my final volume is 20 µl

[ashu2007]

Ozeir Kazemi, on 01 May 2012 - 03:26 PM, said:

for cdna concentration of real-time pcr reaction i used concentration ranging from 50ng-200ng with different primer concentration 1/20, 1/15, 1/10 from 100 µM (micro-molar) stock solution. i'm using nuclease free water not RNAse free water,( does this make any problem?) and i got ct value32.98, 35.48, 33.92 for my three negative controls ( using nuclease free water+ control buffer 1x+primer) and got ct value 9.5( 50 ng cDNA with 50 µM primer) and 9.7(50 ng cDNA with 50 µM primer) which are so strange and the rest of the samples including also my housekeeping gene( GAPDH) didn't reach the threshold line. my final volume is 20 µl

Hi,

I am in the same boat, for me also it doesn't working. I am able to see bands when I am doing normal RT PCR. In case of qRT PCR, when I ran the sample on gel, I am able to see those bands which was present in normal RT PCR.

I think there is no primer dimer in my case, because I am able to see just one band in my RT PCR as well as in qRT PCR.

I am using the same concentration and same volume of primer for qRT PCR as I was using for normal RT PCR.

I am using SYBR Green with 10X PCR Buffer (5 ul), 25mM MgCl2 (1ul), 25mM dNTP's (1ul), Forward primer 10uM (1ul), Reverse primer 10uM(1ul), cDNA template (2uL) and Taq Polymerase (1uL), and double distilled water (38uL).
In case of SYBR green I kept everything same, except this time double distilled water 38uL and SYBR Green 1uL.

I have tried to use 5X, 3X, 2X, 1X, .1X, .5X, .05X, .01X of SYBR GREEN.

[Lapsang]

My qPCR reactions in 384 well plates are generally:

1uM forward primer = 1ul
1uM reverse primer = 1ul
2X Roche LightCycler 480 SYBR Green master mix = 4ul
cDNA (diluted 1 in 15 from reaction made with 3ug RNA)

An average run for me looks something like:

5 minutes at 95°C

45 cycles of:
- 5 seconds at 95°C
- 10 seconds at 60°C
- 20 seconds at 72°C
- 1 second at 81°C (detection)

(+melting curve)