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Insert size longer than expected on PCR - (Feb/07/2006 )

hi
I tried to clone a 483bp gene itnto pet 28, after tow days I got few colonies on the plate, and I just tested them by colony pcr and they gave positve band but, the product was approximately 25bp more than expected on a agorose gel in comparison with marker, is it because of the dmso in the pcr mixture or it is non psecific amplificatiomn? I tested the primers and they are very specific to that gene , what casued the shigt in the band length.
help me ouut
shashi

-shashi-

It is hard to tell by PCR that the insert is really longer than expected. You can sequence the plasmid to confirm.

-pcrman-

hi
i agree with pcrman. 25bp is too little to tell about the length. Do a restriction analysis or a sequencing.

-fred_33-

Sequencing would be the appropriate to check your insert . Restriction digestion also often give a correct picture if there is any change in restriction site due to mutation .best of luck!!!!

Saikat Chakraborty
Mie University
Japan

-SAIKAT-