you might have some nucleases contaminating your DNA. It is also possible that you might have something inhibiting your PCR reactions.

Considering that you are using a non kit DNa purification protocol, the most common contaminants that inhibit the PCR after a DNA extracions are:

-phenol carryover if you perform an organic extraction prior to precipitation

-salts if the wash with ethanol 70% was insufficient

-ethanol remains after the ethanol was if the pellet was not completeley dry

If you could describe wich protocoll you are using we might be able to help.

1. If you are dissolving your extracted DNA in water then try to dissolve in TE buffer as it helps to maintain pH balance of DNA.

2. Phenol is a important hydrocarbon which can destroy your all efforts if it is not properly removed during DNA isolation process. So, be careful...

3. Dont vigorously shake or votex too much, as it breaks DNA sometimes.

DNA is a very stable molecule. The most common source of accelerated degradation is nuclease contamination. Most of bacterial agents can produce nuclease that can digest your DNA. Make sure that the samples are stored under sterilized conditions and in a fast frozen machine. Do not keep your sample at room temperature for long periods, that will reduce bacterial activity if present.

That depends greatly on the extraction protocol that you're using. if you are using a commercial kit, you should not have problems of degradation, provided that stores the DNA correctly (at -20°C can be store for years). If you use a conventional extraction procedure (like phenol/chloroform) and you know, in advance that you want store the DNA during a short period (without freezing), is more appropriate interrupt the isolation in the precipitation step. The DNA can be stored in ethanol for several months even at 4°C without risk of degradation.

possible reasons could be

1- DNAse contamination.(prefer TE buffer than water if for long term storage )

2- low concentration.

3- pararetro behaviour of DNA

4- phenolic contamination (consider using 1% pvp in dna extraction buffer)

Thanks all for your kind suggestions ......I am storing my DNA in TE buffer only.....and I am using salting method for DNA isolation......I get good bands the same day but if i perform gel electrophoresis next day I get smearing.....As Ruth Nicholas said if I do it fresh I get good bands but not PCR.

And I am autoclaving everything except reagents...tips, tubes..

If you don't autoclave reagents you might have a contamination in them. Solutions such as TE should be autoclaved too.

Check the autoclave if it's working properly.

Check the pH of the TE buffer (if too low (around pH 4) it might lead to hydrolysis.

Along with many plausible reason explained above, one reason could be the precipitation of salt during your final precipitation. If you are using chilled Isopropanol, it may give a higher salt precipitation which could be reduced by using it at room temperature.

I would susggest you to wash your plasmid with 100% ethanol insted of isopropanol and dissolved in in TE buffer of pH 7.5 and stored in-20○ C.