Insert size longer than expected on PCR - (Feb/07/2006 )
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
It is hard to tell by PCR that the insert is really longer than expected. You can sequence the plasmid to confirm.
i agree with pcrman. 25bp is too little to tell about the length. Do a restriction analysis or a sequencing.
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!!!!