pcr smear

[bhaktamehul]

Hi,
i want to know what are the causes of pcr smear. coz i have been having some smear from last one week , but early it workd great... i even tried new dna samples, made changes in annealing temp and time and also used same primer from other lab(where it is working),,

so i just want to know what can be the causes of smearing , so that i can sort out problem...

mehul

[danfive]

Most likely rRNA contamination, or too much DNA in PCR, if the smear is at low weight it may be primer-dimer (fix by lowering primer conc).

[bhaktamehul]

no its not primer dimer coz smear starts right from well... dna conc. is about 5ng/ml ... i also tried some other primers today and they worked... by the way i m having problems with Mi23primer..., i even made new dilution for every thing... i can't think of anything else....

[arammik]

Dear bhaktamehul

PCR smears can be caused due to a number of reasons. Template inhibition is one of the reasons, as stated by danfive. When conducting reamplification from a previous product, it is possible to get short smears. Reducing template concentration in serial dilutions might solve the problem. Also, reducing the number of cycles could help; reducing the total amplification also will reduce the non-specific amplification.

Also remember, the same PCR reaction will behave differently from well to well, heating block to heating block, PCR machine to PCR machine and PCR make to PCR make. So keep those things in mind while optimizing your reaction.

When countering PCR contamination, also a reason for smears, try those reagents which are introduced in larger volumes first. Water is an important source of contamination. Change the PCR buffer, MgCl2 solutions and try again. Always remember to thaw-vortex your reagents well before use, and remember to change one variable at a time.

[bhaktamehul]

thanx for replying arammik ... welll i already tried diff dilution for dna sample currently i m running 5ng/ml and i add 2mL dna in my pcr mix(total 10)... but i still have smear.... i use 30 cycles ... so i;ll just try to reduce it to 25 or someting....well today i ran a pcr mix that was prepared by a diff person in a diff lab with all his mix only thing common was the dna samples , pcr machine and electro machine... but still i got only smears... but the funny thing is when that same guy ran the same primer with his dna and his pcr and electrop machine he got amplification... and this thing is really confusing mee....newway thx for replying once again...

[arammik]

Dear bhaktamehul

Yes, I am familiar with this. It often happens that different PCR machines give different results. However One important thing that i would like to tell you. I dont know the reason for this occurence, but when we worked with some genes such as cry in Bacillus thuringiensis. Freshly isolated DNA gave clear bands. But 5 month old DNA (stored at 4oC) gave no amplification or numerous misamplification for the same PCR machine and same stocks. Reisolated DNA again, gave good results. I dont know why it happens, maybe its just messing with our heads. But it does happen. Try using freshly isolated DNA. When everything's going wrong, it is wise to assume everthing that we are doing to be wrong, and try everything out from the scratch - Just to be sure. You'll also learn a lot in the process!!

[bhaktamehul]

thank u agian.. guess what u r absoulutly right... i figure out he prob yesterday .... well i gave my dna sample to this other lab...who r working on same primer... so they tried their dna and my dna... and woola ... my dna didn't ampliefied a lill bit and their dna did really good... therez something wrong with the dna coz it works with other primers... nway i m goin to extract new dna and let u know i get amplification or not... thanx for helping me...
byee

[arammik]

Yea dude,

I just hope it works out, I know how irritating it is not to get results in PCR..hehe

Lemme know if you came through with what you want.

TC

[saly]

smear may be means som hydrolysis

[bhaktamehul]

thanx sally ...