Is the insert not complete? - Sequencing Cloned PCR product (Jun/16/2006 )
I've cloned a PCR product, from degenerate primers, in a pGEM T-easy vector.
I sent it for sequencing and started to look at the results..
I se both sides of the plasmid-all of the insert is sequenced, the beginning of the insert matches one of the primer sequences BUT the second primer sequence (reverse compl) is missing..??
Please, help me understand what is happening..
I've had the exact same end of my insert 2 times though used two different primers in this end!
Is the PCR fragment fragile in some way so that the end fall of. I don't know the exact expected size..
Is the signal (peaks) dropping off when u come to the end of the insert or is there some other sequence corresponding to the vector ?
I'm not sure I understand..
but, The quality of the sequence is OK over the insert and about 30 bases after (on the vector) the peaks in the last third part is much lower and less 'sharp' than in the upper part (but still considered OK by the sequencing company (MWG))
'is there some other sequence corresponding to the vector ?'
in my insert? (I don´t know my sequence, using degenerate primers from other species sequences (fishes)
this is the first insert I'm sequencing--- I appreciate your help!!
The first sequence of ur insert is fine except that the 2nd primer sequence is missing, which means u dont have the entire insert sequence right?
Is the region of the 2nd primer rich in GC?
My 2nd primer is..
CGT TCA T(GT)(GC) T(CT)C TCA TCT
i.e, a mix of 8 primers choosen from alignment of other species
I am not sure whats happening with ur sequencing. Sometimes if the region is GC rich or is forming some weird tertiary structures, then the sequence signal drops off.