PCR trouble shooting
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PCR trouble shooting
A nice troubleshooting guide on PCR reaction including a detailed PCR troubleshooting flowchart and many useful tips and problem-solving suggestions. (Department of Biology, University of Oslo)
Some possible reasons on an unsuccessful PCR:
A. Pilot error hypothesis
B. Template dilution hypothesis
C. Temperature errors hypothesis
D. Unique template hypothesis
E. Buffer problems hypothesis
F. Bad dNTPs hypothesis
G. Bad primers hypothesis
H. Bad enzyme hypothesis
I. Bad karma hypothesis
Last update 11-Apr-2007, Rating Poor of 39 votes.
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Hi! In my PCR i can`t find the desire bands. I`m tring to amplify one region of the serotonin transporter, but this region have a lot of CG, than in pcr i think they form a hairpin! Anybody can help? I already tried BSA, DMSO, and diferent denaturation time.
Thanks!
Rating: n/a
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i am getting single band of my desired product in agarose..
i want to do SSCP
but in PAGE of pcr product as such without denaturing i am getting multiple bands below my desired band ...in such case i couldnt run SSCP PAGE....
so please suggest how to eliminate those nonspecific bands...
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I have a problem in amplifying a 3/5 kb fragment. I design my primers based on alignment result of some plants. My pcr amplify once, then it doesn't work in the same condition in the other times. I had use tempreature, Mg, primer and DNA gradient as well. but my problem don't solve till now.
What's your ideas about it.
Rating: Very Good
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i am getting no band in the CP region of the HBV, i tried on the 25 samples , what would be the reason , please sort it out,
Rating: Poor
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Saish, if u got the product one time and not getting the reproducibility in the result then it may be due to any component of master mix. spin the contents i.e. buffer, dNTPs, primers indiviually and then add. change dNTPs. dont thaw dNTPs again and again. further if u face the same problem then u also try temperature gradient. also specify ur problem.
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hi i am also facing the similar problem of not getting different result every time on doing PCR for the same gene in the same sample (cDNA). plz help.
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Alzika, try temperature gradient for annealing temperature. difference of temperature should be more than 1 degree C. hope u will get the product at temperature around the exact annealing temperature.
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Alzika, dilute DNA and try temperature gradient for annealing. temperature diffrence should be 1 degree C minimum. hope u will get the desired band at any of the temperature around the accurate annealing temperature.
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hi everyone, im amplifying an ITS region (16S-23S) for my lactobacillus strains but im getting two bands instead of three bands.what could be the problem?
Rating: Fair
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Hey everybody
i have been screening for some primers but i can get a sharp band, if i increase the concentration of magnicium cloride or dNTPs, i get non-specific amplification. I normally use a touchdown programme. if i add BSA still i cant get sharp band. what should i do
Rating: Fair
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Hi medhavi, ur DNA may not be pure. due to some impurities or inhibitors It will not give amplification. so either purifiy it or use BSA in PCR reaction. also try other method for DNA extraction. thier should be no greenish colour due to chlorophyl.
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clear bands seen after extraction of plant genomic DNA but after 35 cyclecs of PCR no band is seen
Rating: Fair
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Hi,
I am getting the product of desired size but the band is very faint.... I have tried with decreased n increased annealing temperature but no effect. What should I do to increase the yield.
Thanks
Nirmala
Rating: n/a
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Hi,
I am getting of desired size but the band is very faint.... I have tried with decreased n increased annealing temperature but no effect. What should I do to increase the yield.
Thanks
Nirmala
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Hi everybody,
please I need your help. I'm doing PCR but I'm getting different band intensities for the same gene , each sample is in duplicate, but there is wide differrence between two.So I can't rely on the data. Could you please help me?
thanks
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I am trying to amplify a 395bp product thru' nested PCR but there is one extra band (300bp) coming just below the specific band in all the samples. And the intensity of that band is very specific to the intensity of the band of interest in all the samples. What should i do???
Rating: Good
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Dilute your DNA sample 20 times and try to do PCR again...better to keep denaturation temp around 95.0
Rating: Good
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I am trying to amplify a 300bp fragment using PCR. But I always got smear no clear band. I run two round PCR: first round annealing T is 50 35 cycles, and dilute first round PCR using as templete to run second round with annealing T 55 35 cycles.
What's wrong with my PCR
Thanks
Rating: n/a
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i am not getting band of my choice while doing pcr. earlier i did decreased the annealing temperature but i did get poor band along with a strong inspecific band & some other bands? does it has something to do wth contamination or it is due to unspecific amplification?
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i am trying to amplify 792 bp fragment. But i am getting non specific band of lower than expected size.
Cycle condition are;
1- 94 , 5 min
2- 94, 45 sec
3- 52 , 45 sec
4- 72 , 1 min
5-2-3: 30 cycle
6-72, 10 min
Rating: Good
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I am trying to amplify a 500 bp fragment Using RT-PCR. I am not getting any amplification and what i do get is a faint band which lies far below the running point of the dye. Tm of the primers are 71.0 and 68.0.
Rating: Good
Rating: Poor
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I am trying amplify 1700 pb fragment using RT-PCR .I get this product .but in my next experimet
I dont get this product.please help me to solve this problem.
pcr program:
1- 94 , 3 min
2- 94, 30 sec
3- 57 , 25 sec
4- 72 , 2 min
5-2-3: 35 cycle
6-72,10 min
Rating: Good
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the only thing that I can think about is to try to increase the annealing temperature 1-2 degrees and see if this nonspecific amplification run away.
Rating: n/a
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if your problem from undenaturation you can try to use DMSO or glycerol.
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hii rashmi,
Change the buffer concentration and you primer length, if possible increase your primer lenght about 5 nt.
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hi,
i am doing RAPD pcr from chicken blood. ruunig through 2% agarose gel, i am getting bands but its of seggregated one, very low intensity bands.i have alterd the master mix still the same problem continued. i tried with altering (decreased from 35 to 34)annealing temp seggregation stopped but intensity of band is low. please advice me to solve this problem,.
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i need to have a single sharp band to sequence but instead i get a single sharp band cover in smear, please help me to clear the band and smearless it
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I'm genotyping from mouse genomic DNA. The PCR reaction used to work, but now I'm getting background bands in my wild-type reaction and a smear in my null reaction. The reaction used to be a crisp, single band (1000bp for wt, & 500bp for null). I've even tried using old DNA template that I know has worked and still getting the same results. I've changed all my solutions, and even tried a different thermocycler. Please help!!!
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i have extracted dna of bovine herpes virus-1 from semen samples of bulls. i have tried many methods of dna extraction even the qiagen dneasy kit but failed to extract the good quantity as well as a good ratio of dna. on doing pcr using hot start taq polymerase i hadn't got desired product band even in the control. now i m doing pcr using fermentas master mix. i have tried denaturation at 99.9 degree celcius followed by snap chill and then the normal protocol of pcr with 36 cycles.but now im getting bands in control but not in samples instead smear in is there always in samples as well as in control but band can be visualised in control.plz help me with possible solutions.
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We did PCR with RAPD primers, it seems that the bands stands adjacent with the lanes and did not migrate, but 1kb+ ladder migrated well. We used 1.5% agarose gel.Please suggest us.
Rating: Good
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Result Pages: 1
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