Real Time troubleshooting

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Sushma Dagar
Sushma Dagar's picture
Real Time troubleshooting

I am beginner in Real Time PCR. For doing real time, I am using Syber Green. I am facing problem with standard. For making standard, I am diluting my samples 10 times (1000, 100, 10). but still in all the dilutions, amplication is starting at the same cycle number. &nbsp;Please if anyone can suggest me something.<br /> <br /> Thanks&nbsp;<br /> <br type="_moz" />

morkurbaga
morkurbaga's picture
 Maybe your cDNA

 Maybe your cDNA concentration is too much. So you cannot see the difference between the dilutions? You can do more dilutions and try again? I dont know  I am also new in RTPCR and I have questions waiting for replies :) 

Sushma Dagar
Sushma Dagar's picture
 Thanks for your reply.

 Thanks for your reply. Recently I have tried a range of dilutions from 1000 to 5 but still got the same result.

hcheung
hcheung's picture
What is the cycle number that

What is the cycle number that they are starting at?
If the Ct is >30, then your cDNA is probably too dilute.
If the Ct is <15, then your cDNA is too concentrated.

Sushma Dagar
Sushma Dagar's picture
 They are starting at around

 They are starting at around 20-22 cycle number. but the problem is that highly diluted sample is also starting amplification at almost same cycle number.

wilsonj826
wilsonj826's picture
Hi,

Hi,
You may find this article helpful for your work.
http://www.premierbiosoft.com/tech_notes/real_time_PCR.html

Cheers !!
John
 

Sushma Dagar
Sushma Dagar's picture
 Hey John,

 Hey John,

Thanks for the link