Sanger Sequencing result shows my insert sequence is perfectly right but, as being said in the question, the vector sequence is a mess. Not only is it about a few random substitution or deletion of a few bases, the whole stretch of sequence is just not right.
Before sending for Sanger sequencing, I have digested the miniprep of the subcloning product by the designated restriction enzymes to check whether it gives the linearized vector and the target insert. And the digestion result is alright shown by gel electrophoresis.
However, seeing from the Sanger sequencing result, not even the restriction enzyme site sequence is aligned back to the reference sequence.
Regarding the sequencing primer design: They are priming sites ~60nt away from the insert. Complementary to the vector sequence flanking the cloning sites.
Has any of you encountered similar problems before?
P.S. For alignment, I only chose sequencing result that consists of sharp peaks.
Hi there,
efficient sequence reading is efficient from around 50 to 100 nucleotides downstream the priming sequence. In your case the reading of the plasmid sequence is erratic probably because too close from the primer.
In which vector you have done cloning? Was orientation ok as desired? Keep in mind that good quality sequences will only appear after 70-100bp downstream to priming site as due to too many dideoxy stopped short fragments they are difficult to resolve in capillary and crowding effect is eliminated by software that makes a basline for dye signal strength..
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