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Sequence size and insert size - Sequence size has larger base pair than insert size (Sep/27/2010 )

Hi all,

I did PCR amplification, cloning, and sequencing. My primers for PCR are 27f and 1492r. So, i would expect the amplicon size is 1492-27= 1465 bp. (I wonder is this the right way?)

If this is so, the expected amplicon size is 1465bp.

I then purify the PCR product and proceeded with TOPO cloning.

After selection of positives clones, plasmid is extracted and sent for sequencing.

After discarding the vector sequences (according to the vector map)flanking my insert and done with the alignment using Bioedit,the size of the sequence that suppose to be about 1465bp turns out to be about 1530bp.

I am confused on why my insert size (bp) is not around my amplicon size from PCR? Looking forward for suggestions! Thanks a bunch!



Put your primers into the Primer BLAST and check with refSeq RNA and Genome databases for possible products, the aplication will tell you the teoretical length based on known sequences. Or you can BLAST your sequencing result to see what it matches. Maybe it's just wrong calculation, but it can be differently spliced transcript or insertion.


Are those primers targeting the 16s rRNA gene? Usually, the base positions used in the primer name refer to the positions on a particular strain of E. coli. There is variation in the 16s genes of other species, or even between the multiple 16s rRNA genes on a single chromosome.