Primer dimer issue in real time PCR - (Jun/22/2009 )

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they're dilutions of DNA pool...the curve looks like i forgot to put in the standard dilution and only have primer mix in the wells, but i really did make sure i put everything in...the NTC also gives amplification (with similar curve). i really wonder why none of the primer binds correctly in the standard dilution wells, but able to do so in the wells with samples....

-ctfazra-

ctfazra on Wed Mar 21 12:55:16 2012 said:

Hi,

I'm very new to qPCR, n I saw this thread so I'd just add in my question

I'm using SYBR green to detect various genes (using total RNA), housekeeping (GAPDH) seems to give good results but the problem is the other few genes they keep giving me funny results, i.e. slope > -2.0 though I managed to get R^2 ~ 0.95. After some readings I figured out I should've done the melting curve first to detect primer-dimers formation, so I did.

Attached example here is the melting curve for one gene. Due to the funny results after few attempts previously, I chucked the primer and use the new stock for this run. The samples mostly give Tm~82.5degC, but all standards have lower Tm ~72.5degC. I supposed the lower-Tm amplicons are primer-dimers, but how come the samples do amplify correctly, but none of the standards? What could be the reasons for this, and how do I improve my qPCR?

Thnx

-ctfazra-

Hi,

Can you please help me in qRT-PCR. I am also using SYBR GReen, but I am not able to see the melting curve like yours. Though in my normal RT-PCR I am able to see just one band, which is of my gene of interest.

For normal RT PCR I was using
Double distilled autoclaved water:37uL
10XPCR biuffer: 5uL
25mM MgCL2: 2uL
25mM dNTP's: 1uL
Forward primer:1uL
reverse Primer:1 uL
cDNA: 2uL
Taq Polymerase:1uL

While in case of qRT PCR I was using :

Double distilled autoclaved water:36uL
10XPCR biuffer: 5uL
25mM MgCL2: 2uL
25mM dNTP's: 1uL
Forward primer:1uL
reverse Primer:1 uL
cDNA: 2uL
Taq Polymerase:1uL
SYBR green : 1uL

I tried to use different dilutions of cDNA like 1:10, 1:100 to figure out, but it didn't work. Then I made different dilution of SYBR green 50X, 5X, 3X, 2X, 1X, .1X, .5X, .01X, .05X but not able to see result. However, when I ran the product of qRT PCR into gel I was able to see just one band, which was of the size I required.

Please help me out.

-ashu2007-

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