Primer Dimer problems in real-time PCR-Real-Time PCR

Hi, everyone, I am newcomer and have started Real Time PCR for 2 months. After all, I have designed the primers sets again using Primer Express 1.0 and using Oligo-4.0 to check if there are suspected dimer formation. However, most of primers designed by Primer Express seem to have many dimer formation by upper or lower primers themselves or Upper-lower dimers. So what I can do is only choosing the set with smallest no, of dimer.So I have performed the normal PCR again and run gel to see if it is OK. The samples with my cDNA of coz has the band ~100bp. However, I am so frustrated that the NTC (without cDNA) also has the bands with the same size!!! Aren't primer dimers about 40-50bp only? Also, why it has the same size (~100bp) as the one with cDNA?Do u all have some good suggestions for primer design for Real Time PCR? -lee_liam-

hi first you can use many softwares for designing primers (you can check my previous participations)anyway, the contamination of your RNA samples is still a factor ,another factor is the integrity of your RNA samples ,did you check it with a bioanalyser,also the annealing temperature of your procedure seems to be the best thing to play with right now ,if you have your two primer Tm's close to each other you can lower the temperature safely by one or two degree to make only ideal annealing to happens good luck

-husam-

Hi, am doing Real Time PCR using SYBR green for TRAP (Telomerase) product. I have been getting amplification even in negative controls!(CHECK THE PICTURE) I doubt this is because of primer-dimer formation. I need suggetsions in reducing this during my PCRs...Waiting for suggestions. (picture: green line is +ve all other -ve??)

-Molonco-

QUOTE(lee_liam @ Nov 22 2005, 08:06 PM) [snapback]32075[/snapback] Hi, everyone, I am newcomer and have started Real Time PCR for 2 months. After all, I have designed the primers sets again using Primer Express 1.0 and using Oligo-4.0 to check if there are suspected dimer formation. However, most of primers designed by Primer Express seem to have many dimer formation by upper or lower primers themselves or Upper-lower dimers. So what I can do is only choosing the set with smallest no, of dimer. So I have performed the normal PCR again and run gel to see if it is OK. The samples with my cDNA of coz has the band ~100bp. However, I am so frustrated that the NTC (without cDNA) also has the bands with the same size!!! Aren't primer dimers about 40-50bp only? Also, why it has the same size (~100bp) as the one with cDNA? Do u all have some good suggestions for primer design for Real Time PCR? Dimer formation by upper primer itself - means that when you add just one primer you get a dimer forming? If this is what you mean then it's an odd result.'Primer dimer' can be any size theoretically but I imagine that there is competition between different forms and the shorter ones tend to win out and it's those ones that are seen in the end.What size would the product be if genomic dna had served as target. Or are pcrs designed across intron/exon boundaries so genomic dna not a problem? -tiredkhan-

QUOTE(tiredkhan @ Nov 26 2005, 09:22 AM) [snapback]32528[/snapback] QUOTE(lee_liam @ Nov 22 2005, 08:06 PM) [snapback]32075[/snapback] Hi, everyone, I am newcomer and have started Real Time PCR for 2 months. After all, I have designed the primers sets again using Primer Express 1.0 and using Oligo-4.0 to check if there are suspected dimer formation. However, most of primers designed by Primer Express seem to have many dimer formation by upper or lower primers themselves or Upper-lower dimers. So what I can do is only choosing the set with smallest no, of dimer. So I have performed the normal PCR again and run gel to see if it is OK. The samples with my cDNA of coz has the band ~100bp. However, I am so frustrated that the NTC (without cDNA) also has the bands with the same size!!! Aren't primer dimers about 40-50bp only? Also, why it has the same size (~100bp) as the one with cDNA? Do u all have some good suggestions for primer design for Real Time PCR? Dimer formation by upper primer itself - means that when you add just one primer you get a dimer forming? If this is what you mean then it's an odd result. 'Primer dimer' can be any size theoretically but I imagine that there is competition between different forms and the shorter ones tend to win out and it's those ones that are seen in the end. What size would the product be if genomic dna had served as target. Or are pcrs designed across intron/exon boundaries so genomic dna not a problem? Thx u1 IN fact, what I means thhe dimer formation of "upper primer" is that after I have checked the Oligo-4.0, the upper primer also seems to have dimer themselves.For the template, I have used the RNA instead of genomic DNA and using the ABI reverse transcription reagent in order to make the total cDNA using random hexamer as primers -lee_liam-

QUOTE(lee_liam @ Nov 26 2005, 03:51 PM) [snapback]32537[/snapback] QUOTE(tiredkhan @ Nov 26 2005, 09:22 AM) [snapback]32528[/snapback] QUOTE(lee_liam @ Nov 22 2005, 08:06 PM) [snapback]32075[/snapback] Hi, everyone, I am newcomer and have started Real Time PCR for 2 months. After all, I have designed the primers sets again using Primer Express 1.0 and using Oligo-4.0 to check if there are suspected dimer formation. However, most of primers designed by Primer Express seem to have many dimer formation by upper or lower primers themselves or Upper-lower dimers. So what I can do is only choosing the set with smallest no, of dimer. So I have performed the normal PCR again and run gel to see if it is OK. The samples with my cDNA of coz has the band ~100bp. However, I am so frustrated that the NTC (without cDNA) also has the bands with the same size!!! Aren't primer dimers about 40-50bp only? Also, why it has the same size (~100bp) as the one with cDNA? Do u all have some good suggestions for primer design for Real Time PCR? Dimer formation by upper primer itself - means that when you add just one primer you get a dimer forming? If this is what you mean then it's an odd result. 'Primer dimer' can be any size theoretically but I imagine that there is competition between different forms and the shorter ones tend to win out and it's those ones that are seen in the end. What size would the product be if genomic dna had served as target. Or are pcrs designed across intron/exon boundaries so genomic dna not a problem? Thx u1 IN fact, what I means thhe dimer formation of "upper primer" is that after I have checked the Oligo-4.0, the upper primer also seems to have dimer themselves. For the template, I have used the RNA instead of genomic DNA and using the ABI reverse transcription reagent in order to make the total cDNA using random hexamer as primers I'd be interested in other people's opinions but seems to me that primer dimer involving just one primer is not normally a problem so you should concentrate on avoiding particular pairs of primers that might cause problems. This is because the initial mispriming events are relatively rare, and for a single primer would give some sort of panhandle structure that won't be amplifiable. So you would just lose a certain fraction of your primer every cycle. This is why it's sometimes useful to add more of one primer (the one that you are gradually losing) than the other.Mispriming between 2 different primers is also usually a rare event but can give something that is not only amplifiable but often amplified more efficiently than the thing you want so can be a big problem. -tiredkhan-

wow, yeah, that's a problem do you have a pic of your melting curves you could attach? Also, from the Tm, you can determine if it is probably a primer-dimer. if it is not, you have contamination. there are many websites devoted to reducing contamination in both PCR and when working with RNA. I would recommend Ambion, Promega, NEB, ABI....

-aimikins-

i would say a single primer forming a dimer would be a hairpin, and those are definitely to be avoided; the two homologous sequences are already close together and if they bind with good affinity there will be no product  

-aimikins-

Hi, you can check the melt curve to judge whether there form primer-dimer,or run product of real time PCR to check this on gel elect. the backgroud of some the tube is high, the effeciency of the reaction is low, analysis the slope and effeciency to decide the primer and the reaction condition is whether favoriate.

-dixifor-

its possible to have primer dimers when there is no template or less template. To check this out you have to do a serial dilution of the template (10 fold would be good) and then rule out this possibility (less/no template). And in future expt. you would be doing the PCR at required template concentration. The other possibility is that you have the eternal problem of PCR contamination. Try to rule out that problem also, may be by using some standard primer which your lab or any one you know of. I hope this will help you. And lastly you can use Primer databank to ｓｅｌｅｃｔ primers for your gene of interest as they either published or tested.

-srisashi-

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