Real time PCR late amplification - (May/10/2006 )
Dear all,
I am doing realtime PCR on the Corbett Rotor Gene 3000. I use actin (approx. 500bp product) as a housekeeping gene to produce a standard curve from cDNA (concentration 31.5ug/ul in 25ul). I use SYBR Green Mix from Qiagen. I know the product size is a little too big...
Out of 6 times I did a 10X serial dilution, only once I got a reasonably nice standard curve. But the amplification curve began late (around 35 cycles). When I tried to repeat exactly the same conditions, the amplification curves began only after 45 cycles and very little fluorescence <15Fl was detected. Melt curves are ok though.
What is the problem? Please help!! 
Many thanks 
I am doing realtime PCR on the Corbett Rotor Gene 3000. I use actin (approx. 500bp product) as a housekeeping gene to produce a standard curve from cDNA (concentration 31.5ug/ul in 25ul). I use SYBR Green Mix from Qiagen. I know the product size is a little too big...
Out of 6 times I did a 10X serial dilution, only once I got a reasonably nice standard curve. But the amplification curve began late (around 35 cycles). When I tried to repeat exactly the same conditions, the amplification curves began only after 45 cycles and very little fluorescence <15Fl was detected. Melt curves are ok though.
What is the problem? Please help!!
Many thanks
dear Chris,
You may run your PCR product on gel, just making sure that your PCR reaction is working.
If you dont see a band, optimized your PCR.
Best regards
I am doing realtime PCR on the Corbett Rotor Gene 3000. I use actin (approx. 500bp product) as a housekeeping gene to produce a standard curve from cDNA (concentration 31.5ug/ul in 25ul). I use SYBR Green Mix from Qiagen. I know the product size is a little too big...
Out of 6 times I did a 10X serial dilution, only once I got a reasonably nice standard curve. But the amplification curve began late (around 35 cycles). When I tried to repeat exactly the same conditions, the amplification curves began only after 45 cycles and very little fluorescence <15Fl was detected. Melt curves are ok though.
What is the problem? Please help!!
Many thanks
Hi Chris,
I`m also using the Rotor Gene for my real time experiments. I have amplified from 100 to 600 bp PCR products. Based on what I`ve seen, the efficiency of amplification does go down as your product increases beyond 400 bp. Couldn`t you just design an internal primer (you only need one) to amplify a shorter fragment? If not, you can increase your hkg template concentration if you want your CTs to be in the lower range but then again, maybe this (higher concentration) is not what you want for your target gene. Goodluck.
Casandra
Hi!
Thanks for the suggestions. Run on gel shows the product. I can't design another primer coz I am verifying results for semi-quantitative reverse-transcriptase PCR. So I would like to maintain all primers and conditions as best as I can. That's why I am having all sorts of problems in optimizing my Real time.
What it the use of GAIN in a realtime program? Is it important in the PCR itself? coz i dunno about gains i juz auto calibrate it. Range from gain 3-7. My cycling conditions are 95C 15 min (HotStarTaq), 94C 20 sec, Annealing 30 sec, 72C 30 sec. Does the cycling time and number of cycles have anything to do with the amplification efficiency? Is amplification after 35 cycles acceptable as a valid result? Coz my senior heard after 25 cycles its not acceptable coz considered as unspecific amplification.
Many thanks
Chris.
Chris-
How are you measuring or determining your amounts of cDNA? Are you overestimating your cDNA concentration? Maybe your samples are more dilute than you think?
I agree that amplification after 35 cycles is generally not reliable. Anything after 35 usually is in the single copy range of cDNA.
I use biophotometer. Dilution 1 in 49. What do you mean single copy cDNA? SYBR Green can't bind to ssDNA right?
Now I'm suspecting the machine has a problem as my colleague is facing the same problem. I think there's something wrong with the detector for the SYBR green. Has anyone faced this problem before?
Chris
Hi Chris-
Sorry, I don't mean single-stranded cDNA, I mean that any signal you see that late probably started from only a few copies of original template, and/or non-specific template.
Sorry, I can't help you with the detector problem!
I don't know how a biophotometer works, so I guess I'm stuck there, too. I just wanted to make sure you weren't measuring cDNA at A260 after your RT reaction.