Primers work for regulary PCR but not real-time PCR - why? (Aug/26/2006 )

I have designed 4 pairs of primers, which can prime the target product in normal PCR, but cannot work in real-time PCR.


Why?

I designed them using DNA Star, choosing the best choice.

Is there any web site or software good at designing real-time primers?

And, what about the PrimerBank?
Are the primers there efficient?

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Dear Annezhu,
Few question to ask?
1. What real-time PCR machenism are you suing? TaqMan or SYBR Geen? Or..... something else?
2. Are you running 4 pairs seperately or multiplex?
3. Did you set your real-time PCR cycling profile same as normal PCR?

Regards

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Dear Hadrian,

Thanks for your reply, as for your questions:

1. What real-time PCR machenism are you suing? TaqMan or SYBR Geen? Or..... something else?
---- We are using BIO-RAD real-time PCR device. SYBR.

2. Are you running 4 pairs seperately or multiplex?
----I running 4 pairs seperately.

3. Did you set your real-time PCR cycling profile same as normal PCR?
----I set my cycling as follow: 94 oC 15 sec, Anealing 15 sec, 72oC 20sec.

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the conditions for the real time are the same as for the reguar pcr?
dont have to much experience in real time but if you change the extensively, you might not have amplification at all

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Hi annezhu,
I had worked with PCR primers and now QPCR. The cycle condition of PCR is usually different than that of QPCR. Using PCR, sequences mostly longer than 100bp are amplified, however, for QPCR the length is usually restricted to ~100bp. This means the temperature and time for annealing and extension steps have to be adjusted, thus primers for normal PCR are mostly not suitable for QPCR. I use PrimerExpress, this works good. Let's have a try. Hope my answer means somthing for you.

Ribosoul

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Thanks a lot for your help.
I will have a try.

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Good thing to remember

"Mrs PCR" hate all "Mrs RealTimeChemistry" and they do not like to live in one house (tube)

that is why your results are correct and not surprising

Much more surprizing results when "Mrs PCR" amd " Mrs Realtimechem" do not conflict and give something
that look like Results

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1 Biorad Kit or homemade ?
2.Genomic or cDNA ?

Reoptimize input template Mg and cycling using mix with agarose gel detection
Try SYTO9 instead of SYBR

modern CCD detectoin of agarose gel products for example with SybrGold and Blue
transilluminators can detect 25 pg of DNA per band and "background" is spread along gel!

it is Far bellow SYBRREALTIME where product and background is in one tube


that is why you may make simple mistake - regular PCR works but realtime
doesnot - it also works but can not detect

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Thanks very much for your help.

As for the questions:
1 Biorad Kit or homemade ?
---I use BioRad's iQ SYBR Green Supermix.

2.Genomic or cDNA ?
---cDAN.

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