Expected band for PCR smears..

[MicroKiller]

I have recently tried to "fish out" a gene from my total extract. I have designed the primers accordingly and when i run my PCR the band of my expected size is not clearly visible but smears. I have tried changing the annealing temperature hoping for better specificity of the primers, using fresh samples etc.. any ideas or suggetions???

[koukou2]

Hey MicroKiller!

when you mean "fish out" are you trying to PCR it straight from genomic?
or cDNA ? I can help you more if you tell me which as I am generally good with PCR

hmm sound like:

1) maybe your dna is bad quality or degraded? - but you tried fresh samples...maybe the kit is bad? or you eluted with EDTA which messes up PCRs...?

2) Do you have a gradient machine? as you tried many temperatures you probably do... maybe you should try a gradient from 50C - 70C if you havent

3) is your sequence GC rich? smearing all over means probably non specific amplification.... try either making new primers, using DMSO

4) are your primers ok? try checking primer self-annealing using program

5) more to think of soon!

good luck!

[MicroKiller]

hi kou kou,
I started with a total RNA extraction followed by rt-pcr. then i did pcr for the rt product. the primers are not tht high with gc content. any ideas?

[koukou2]

are you trying to PCR the whole cDNA fragment? as this is quite difficult if not impossible for many genes...

(as RNA may be degraded and is not always full length after RT)

have you tried 5'RACE or 3'RACE? to get the whole piece and then pcr?
do you know the full sequence of the gene you are pcring?

[MicroKiller]

I dont know wht whole gene sequence.. and the 5' and 3' RACE.. wht does tht mean? i dont know wht is tht.. i am only starting to work on PCR.. kekeke

[PCR-Man]

Hi MicroKiller,

what are the conditions you are running the gel? %agarose, buffer, voltage blablabla.
And how much do you load on the gel? Does the ladder smear as well?
And what are the PCR conditions? How much MgCl2 what annealing temp, how long, what polymerase, how many cycles., what gene, what organism

;-)

[Topaz2]

Good ideas PCR-MAN!

[MicroKiller]

Everything was optimized according to the recommended concentrations.. i have tried playing with the annealling temp... from low to high.. but i have tried lowering the amount of primers used..... looks like its better.. but i have stopped running the samples.. will continue later..