I am trying to sequence a potential KO by submitting samples to Genewiz, and I am getting this repeat in one of my sequences.
Here is my PCR Steps
-98 degrees for 2 minutes
-Touchdown 7 times
-30 Cycles of the following
-98 degree denature for 10 seconds
-60 degree annealing for 20 seconds
-elongation at 72 degrees for 30 seconds
-72 degrees for 5 minutes-
-end cycle
i am not sure if i need to alter my temperature, or if it is my primers, but I am at a loss. does anyone have any ideas?
Dear MAtthew
is the repeat corresponding to the sequence of one primer?
Because sometimes happen to me that i obtain some clones that contains at the junctions multiple primer sequences. In this cases, i solved the problem just screening a larger number of colonies (only some colony contain this artefact) or repeating the PCR with lower amount of the primers.
good luck
MAnuele
Hi Matthew,
can you first test your primers for specific hybridization on PCR in silico at UCSC (http://genome.ucsc.edu/cgi-bin/hgPcr) or NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and see on a genome browser if variants have been released in your sequence.
fred
In addition to the answers by Manuele Martinelli and Frederic Lepretre you should confirm that the quality of the sequence you are getting is high quality and reliable. Sometimes a bad template will generate "sequence" from the machine but it is really random noise. Without seeing your actual results we really can't accurately assess what is happening.
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