Hello! Many factors could be responsible for your problem. However, you should try running a temperature gradient PCR first, so as to get the optimal temperatures at which non-specificity of your primers would be best eliminated. Secondly, if you are targeting mitochondrial genes such as the cytochrome oxidase genes; you should know that such genes are highly conserved among species. In essence, search for regions of hypervariability while designing your primers. Best of luck!

Hi,

did you design the Primers for gDNA or cDNA?

BR

Daniel

hi, its very common problem or you may say it happens many times even if we repeat a standardized protocol after some time. More frequently it happens in case of genomic DNA.

you have to standardize your PCR condition again.....specially Tm, MgCl2 conc, try MgSO4 in place of MgCl2,

i hope it will give you your band.

you can try eluted product of your desired band as a template for PCR

wishes

Thanks, my DNA is from gram negative bacteria and cheked itis purity before by electrophoresis.

your primer might be having problem, either binding with more than 1 regions... or getting dimerize....... try to increase annealing temperature little bit.. to make more strangest binding with gene of intreats.....

Hi

Its a common problem...

U might try one of the followings:

1) Decreasing MgCl2 conc

2) As ur product is just 460bp, so u cn safely decrease ur extending time to 30 secs.

3) use a range of temperatures on gradient PCR for findind the best Annealing temp

thanks erery body. i want you to be know i used standrd master mix from Bioneer company at my work but i just added my genomic DNA templete 5ul from -ve bacteria and 1.5ul as 10picomolar of each primer.

Dear friends, i used standrd pcr master mix from Bioneer company and i just added 5ul of genomic DNA and 1.5ul of 10 picomolar of each primer then completey into 20ul by pcr water

hey Hassan....Its molecular biology...and you will face such a problem each day..

.i think your problem is not so big..plz try again with the suggestions and i know you will got your band..

in pcr master mix its tough to standardize the concentrations. so i suggest you to use independent components for standardization.

wishes

Yeah dts rite...using independent components is always the method of choice...coz then u can precisely knw wats working n wats not...gives u better control ovr ur reaction..

If you have gradient thermocycler for optimization of Tm as well the MgCl2 concentration.

1st step would be to find your Optimized Tm (between 60 - 75 C)

2nd step is to get MgCl2 concentration optimization by using multiple tubes with 0.5 mM decreasing and increasing MgCl2 reaction

If this dosn't help than probably primer design is faulty. Use Gene Fisher primer design program for it.

Ahsan Sheikh

hey i want to ask you are the primers completely gene specific? if that is not the case then you can get some nonspecific binding and thus multiple bands.

i suggest you to redesign primers from any professional person and they should not have any false primers and dimers. moreover , you should not design any primers that have triple base sequence at the send . this is your possible problem.

Was the primer giving a single band earlier ? If yes, Optimize the PCR conditions as said above. If not, the primers are not specific. As, if the MgCl2 or other problems exists there well not be any amplification; or you may not get proper bands

hi

Reduce the DNA volume. Add 1ul of genomic DNA and make it for 20ul reaction. If you are not getting try gradient PCR to standardize your anneling temperature of primer.

It would suggest to increase the annealing temperature of the reaction, one degree at a time, or simply use gradient PCR. Next thing you can do is to use DMSO which is an enhancer. It specifies the reaction.

Regards

try to do BLAST search for your primers

Its really a common problem. MgCl2 conc and Tm is to be reconsiderd if the primers are specific. Moreover reduction in extension period is to be tested.

I think your thermal protocol is right, but if you 've gotten multiple band for several times on an extracted DNA sample, It may be due to contaminations otherwise you have probably primer dimers as short bands.

Hey Hassan,

Can I ask, what gene region is it that you are trying to amplify? I had the same problem for a while when I was trying to amplify from the ribosomal genes. After sequencing some of the products it appeared that they were actually the same thing and that secondary structures were maybe appearing on the gel! Just a thought :)

Dear Kambiz can you tell precisely which kind of contamination may be there.

Simply, every extracted or non extracted DNAs present in environment could be amplified by your mix if contaminated.

Hi Find your primers in gene by BLAST. You can also try avoiding more GC repeat at 3' end of a primer. Only 2 GC content at 3' end is recommended.

Hello! Many factors could be responsible for your problem. However, you should try running a temperature gradient PCR first, so as to get the optimal temperatures at which non-specificity of your primers would be best eliminated. Secondly, if you are targeting mitochondrial genes such as the cytochrome oxidase genes; you should know that such genes are highly conserved among species. In essence, search for regions of hypervariability while designing your primers. Best of luck!

if you know your ta or you can get it from the software you used for designing primer sequence, then try Touch Down PCR( a temp + 2/3 of your ta and gradually reduce -0.5C/cycle till your respective ta) this might reduce the nonspecific bands

thanks Benedict Odii for the info

Oligo gives me 65.4 and 64.2 degrees for your primers as annealing temperature. I usually use 2 degrees below whatever it gives me - this does depend on your Mg++ concentration of course, but add the non-specificity you observe, and I would definitely say your annealing temperature is too low.

Hi,If the length of extra band is more than 500bp I suggest you try pcr with 30second for extention step because for each 500 nucleotid 30 s is enough!your favourit band is 460bp .in this way, extra bands extention will be prevented.

goof luck

I agree with mthe notion that your primers havenon specific binding properties. why not try other primers designed with more specificity

f.elasmer

Might be the primers are not used up completely and also try for more cycles...so that all the primers are used up.

All the Best

your primer is not work properly so you should first check your primer sequence and for increase their specificity use DMSO @ 1 microlitr for 20 microlitre reaction mixture.

First you check your primers homology with the sequence and through the whole genome of respective organism. go for BLSAT.

moreover you can change your pcr conditions ..... give only 15 seconds for annealing and reduce your cycle number. you aslo go for a gradient pcr with respect to tem and mgcl2. hope this may help you. good luck.

increase your Tm

Hi!

Obviously your primers are not very specific. Did you blast them?

BR

D.

hi..hachim.. tell me one thing r u getting band of ur interest among multiple bands.if yes then increase Tm of your reaction and decrease the time of both annealing as well as extenstion because ur gene is not so long.one thing u can also do that decrease amonunt of Mgcl2 with increase Tm

I am stunned that you use a 1min annealing time, 1 min is too long.

Hi Hassan, may i know whether or not u already solved d problem? cos its already 4 months an by now u should have solved it..if yes can u pls describe what u have done 2 solve the problem...cos it might help others...if not then others can give further suggestions...however...does ur multiple bands includes ur target gene...try 2 use gradient PCR as it will leads 2 the best annealing temp for ur primers...n then u can optimize ur primers as well in order 2 get a very precise result by trying a different concentration of forward n reverse primers..then u try other parameters like MgCl concentration n etc etc...

If you can not perform gradient PCR, increase your annealing tempereture and decrease the extension time to 0,5 min. Otherwise you should optimize your PCR condition with different Mg2+ concentrations and also play with different concentrations of primers and template. Good Luck

1-you can do different Temp annealing start from 50 ,50.5,51.....60

2-confirm your Primer sequence

Good luck

Try to put 5% DMSO to your PCR mixture...good luck...

Hi, try after increasing the annealing temp from 55 to 55.5 and 56, because in extreme stringent conditions only true match will polymerize and spurious product could not. We have done this earlier.

To optimice the primer to

aneling temperature

I have gone through your primers seq, according to the AT2+GC4= Tm formula melting temp of your primers is Forward primer 60 degree C and for Backward primer it is 58 degree C, so there must be something gone wrong while you designed them because there are several faults................ I have analysed your primers both have 2 dimers and 1 internal loops, might be they creating problem...............

1. the Tm difference between Forward and Revrse primers should not be more than 1 degree C, and in your case it is 2 degree C

2. terminal base should be a purine base

3. Primers should be devoid of dimers, hairpins, bulge loops and internal loops have you checked all these secondary structures.

4. And the major factor is you have chosen 55 degree C, annealing temp= Tm - 5, as far the this formula concerned lessening 5 degree C from Tm is right, but the stretches of GC in forward primer probably creating problem, so take it higher.....................means try at 56 degree C.

5. Otherwise as other gentle mans suggest better, if you opt for temp gradient PCR because that might help you better to optimize the actual annealing temp.

6. Have you BLAST your primers, may be they have higher ambiguity and anneal with more than one targets.

But in india the conditions for carryout some other alternatives is not a smooth thing. So better if you try with 56 or 57 degree annealing temp, I am sure that then you must get single band of PCR product.

best of luck

I brief I can say most probably there are two factor which creating this mess..................

one if the non-specificity of your primers towards the target gene

second is the presence of secondary structures in primer seq

now the solution of both is ......................increase your annealing temp, if even then it did not work then got for temp gradient PCR or hot start PCR

Hi

you can reduce your extenting time for hiding of none specific bands.

hi, I think that many factor could be responsible of your problem. I think that your annealing temp could be too low, your primer, have a Tm of 60 C, for increase your specificity you can use it for annealing. Another problem may be the time of annealing, I think that 1 min is too high, ( I think that all your temperature are too high, for 500 pb 30- 40s should be ok! ) this increase the amount of the aspecific product. I would check also the sequence of the forward primer, there are five G and C in 3' , this is not good for the specificity

good luck

You are right Marco

Also if u have this primer for a long time it can make for u this kind of problems.

You can reduce the multiple bands by reducing your template. If your template is a PCR product, then its wise to do template dilution (10-1, 10-2) to see which concentration gives you clean product. Also, check if your primer concentration is too high, typically 0.2uM final is good enough. Also for a small product of 460bp, you can reduce the annealing time to 30 sec and extension time to 45 sec from 1 min. Hope this helps!

Interesting