sequencing problem - (Jan/08/2009 )
I just wanted to ask... if I have sequenced a plasmid DNA using the Universal M13 reverse anf M13 forward primers, i am supposed to obtain complementary reading. Am i correct or wrong. U see i have sequenced the plasmid to isolate microsets but both my forward and reverse sequences are not complementary. I am not very sure on how do i read the electropherogram result and im not sure if i have read and looked at it correctly. this study is actualy using 5' anchored PCR technique. The cloned insert is TG7 repeat and it was found in the reverse sequence. But the forward was not at all complementary. Pls share with me any sites where i can learn on how to read and understand sequencing results, as i am very new to this field. Thank you.
But maybe the insert is too big for the two strands to join and to overlap?
Highly dependent on the size of your insert. Big insert you prolly have to walk it out to get the full complementary/ overlapping sequence.
Insert of bigger than 700bp might not give u full overlapping seq considering the typical good yield of seq is around that size +/- 300bp.