What primer you use? Do you maintain positive and negative control for those reactions? If the primer can amplify a known DNA sample maintain it as a positive control. If you are seeing bands in 20 ul reaction definitely you should get product in 60 ul reactions. If not, then there should be a problem with following:

1. Preparation of Master mix.

2. Settings of your PCR. Check the maximum volume of wells that the PCR has been set. If the PCR is set for lower volume, then there will be a problem in temperature maintenance thereby resulting in erroneous reaction.

3. If you are repeatedly getting products in 20 ul reaction, then prepare a reaction mix for 100 ul and aliquot it into five "20 ul" reactions. Out of 5 tubes, one tube can be used to check the band.

Hope this helps! All the best for your research!

@venkatesh Kumaresan sir...

Thank you for the answer.

Am trying to do the same as you mentioned in last step ...

Let u know the results soon... 

thank you for the support

Besides all possible technical errors: Do you use 94oC - treated bacterial suspension for PCR mix or alive bacteria? Time for bacteria lysis in 60 mkl must be longer comparing to 20 mkl. 

Colony PCR can be tricky. The reason for this is the sometimes poor quality of the colony material (presence of contaminants, genomic DNA, etc.). If you get a negative result from a colony PCR it is often possible that your clone is still positive. It is very important that the colonies you use are freshly grown (that is overnight growth). If possible, always include a positive (e. g. purified plasmid DNA) and a negative control (containing all components except template DNA) in your experimental set-up. A common error is that people use too much colony material. Less is more when it comes to colony PCR.

@Alex Ignatov 

Thank you for the suggestion.

I am using 94oC treated bacteria as template. The bacteria is wild type not cloned one. In my lab  PCR, the highest volume can be set for the 0.2 ml tube 50ul. Is it a problem when I setting 60ul in the 0.2ml tube? before a month I got results from 0.2 ml tube with 60ul reaction with the same reaction setting. 

I planned to set 3 20ul of reaction for same template and let u know the results soon. 

Thank you once again.

@ Christian Q. Scheckhuber

Thank you for the suggestion.  The negative results mean I got only smear after doing in 60 ul reaction though I got high intense band in 20ul reaction. 

Shall I use the 20 ul reaction product as template? I have tried with the same already. 

If  I can use product as template mean how much I shall use as template? please help me in this regard. 

Thank you once again 

@ Muneeswaran Thillaichidambaram

Negative result means that you get no bands. If you see an intense band in the 20 µl negative control reaction (meaning there is no template DNA which you added to the reaction mixture) then you have a DNA contamination in your PCR reaction. 

Using previous PCR products for a second round of amplification is not recommended unless you use primer binding sites that are situated just inside the binding sites for the first round PCR ('nested PCR'). Otherwise you just might amplify some unspecific sequence.

Cheers, Christian