Dear Pradipta

• I dont have direct experience of what you are doing but generally speaking in any assay where you are utilising oligo's that are > 30bps it is best to prepare those oligos with a higher than standard desalting regimen:
• That can be page purified or HPLC for example as you suggest
• In essence, purified oligo's will have a much higher percentage of the full length species you are interested in particualrly when the said oligo is 30bp or above (thus a standard 21bp oligo as implied is perfectly pure and efficiient at standard desalted grade)
• In addition, for more complex manipulations, exemplified by your procedure, the use of high grade (purified) oligo's tends to keep reaction efficiencies at their highest which is particualrly critical ahen attemptingh complex multi step molecular procedure (such as yours)

Agree with @ Laurence Stuart Dawkins-Hall

Thanks dear Hall and dear Mahmoudpour. I have diluted the template for using in lower amount and also I am using PAGE purification for >40ntds oligos.