Hello Dewi

Are you amplifying from genomic DNA, cDNA (etc) ? Having more details might help shed light on the specifics of your problem :-)

Hieee,

You first try an insilico PCR with your primers and check if it gives multiple products.

Also check the Tm you are using. You can try gradient PCR and check if change in Tm can help you.

If nothing works you can redesign your primers (in case if they are too short increase their length to an extant  so that they are very specific)

In the worst case you may extract your amplicon from gel and re amplify it.

Dear Laurence stuart Hall

I used cDNA Mouse Embryonic Fibroblast. 

If it is cdna could it be that you have alternative splicing events? Have you checked if this has been described for the region you are studying? You could also check this possibility excising each band from the gel and sequencing each of them individually.

Dear Sai,

I have already checked by insilico PCR and my gene was.., but my results PCR the band of my gene so weak than unexpected band

A post-PCR solution could be to remove the band from a 3% Agarose Gel. Here is a copy-paste text from an article in review I have just sent to FSI: Genetics.

"The DNA bands were cut with a scalpel from the gel, extracted from the agarose by using the QIAEX II Gel Extraction Kit (Qiagen GmbH, Hilden, Germany), and purified with the MinElute PCR Purification Kit (Qiagen GmbH, Hilden, Germany). After purification the reamplification and sequencing of each allele were performed"

I hope it would be helpful, 

Endika

Dear Dewi

you should try to annealing temp gradient shuold be work .

Dear Herriko

how about etBr percentage you used in Agarose 3%? Thanks

Dear Abid, 

I have done 3 different annealing Tm, but still got unexpected band.

one more things % of agrose gel some time small product did not see propyl. so please you run you product 3 %gel. might be you well get appropriate result. etber concentration is 0.05ug/ml

Hi Dewi, 

my name is not "Herriko". I am Endika. It's a basque name, from the Basque Country, in the northern coast of Spain.

I carried out the agarose gel in a BioRad electrophoretic device with an agarose gel of 3% (by mixing 100 ml TBE 1X with 3 gr of agarose). The migration can take up to 3 hours at 70V (in order to differentiate the bands if they are so close). Then, you have to cut the bands of interest (gel portions), extract them from the agarose and purified (with the mentioned kits) in order to make a new PCR for these fragments (as the recovered quantity is so poor). After that, the amplification only would take place with your region of interest.

If you don't want to buy these kits, which could be so expensive, you can try to stop the migration of the gel and with a scalpel, make a hole just after your band and continue with the migration after that. Now, the DNA of your band should fall down in that hole and you should be capable of aspiring the liquid with your DNA fragments of interest with a pipette. For carrying out this procedure, the TBE1X of the migration device can not exceed the height gel. You can search information about this method on the Internet.

I hope this could solve your problem, 

Endika

Dear Juliana,

You changed your annealing temperature mean you decreased or increased? Yet you can't change Tm of your primer because it is something you can't manipulate as it's the nature of your nucleotides making the primers. To avoid unspecific amplification increase annealing tempreture like 5 degree c. more than Tm. or program your thermo cycler  with temperature gradients so that amplification could happen at optimum annealing temperature. The other option is decrease the con. of MgCl2 so that the specificity of your primer increases to see specific band.

Hello Dewi, 

* In addition to suggestions about the annealing temperature, maybe the number of cycles during the amplification is too high, and according to literature, the increment of cycles may decrease the primers specificity. I would try that one first, before i re-design the primers.  

* If the changes of conditions of your PCR do not eliminate the several unexpected bands, you would have to cut them from an agarose gel, as Endika suggests. One of the kits I have used to purify bands from agarose and acrylamide indicates that the gel has to be agarose 1% (instead of 3%). Also, I think that the 1% agarose gel may help the bands separate better. 

Great luck!

Martha  

Hi Dewi, 

If the band is longer than the band you are looking for, you can also decrease the extension time. Generally, the polymerase you are using will have published extension rates and you can calculate how much time you should be allowing for extension of your band only. Let me know if that does not make sense and I can help you with the calculations!

Renae

About the separation in the agarose gel I don't agree with Martha's answer. 

It's true that you can perform the extraction from a 1% agarose gel if your bands are not very close (have very different sizes).

If not, you need to increase the agarose gel concentration to 2-3%) in order to obtain a better separation between the bands. 

Thanks very much for your suggestion, Is there any possibility my gene is low expressing level? 

Hi Yuliana,

i suggest one (or more) of the following solutions:

• increase the annealing temperature.
• increase time\ temperature of the template denaturation (especially if you have a high genome concentration or complex genome).
• use a PCR enhancer (  DMSO will be fine).
• Check out your template. (high concentration and low purity may cause dimers).
• use high quality Tag (as Pfu)
• check your primers design, use OligoAnalyzer 3.1 (http://eu.idtdna.com/calc/analyzer) for checking your primers.
•  redesign a new primers (primer plus 3 software will be great )

I hope this will be helpful.

Good luck..........

i agreed with Dr. Hayder    and start with templet dilutions    good luck

dilution of sample

i am working in extraction of DNA from pus aspirate for TB, i am facing the same problem but i solve it by sample dilution.