I am having trouble figuring out how my results after FastCloning show that only the template was amplified. I have three sets of primers that are designed to insert anywhere from 10-12 AA's at the C-terminus of a protein in E.coli. I had results from all three sequenced and they were all simply the template. I have had some success with FastCloning in the past, so this isn't my first go at it, but I am a bit confused by this result.
Any advice or guidance is much appreciated!
Brent
Was your no template control also amplifying as well as your samples. You may have pcr contamination in the lab from your previous pcr amplification of this gene, Go back to results from previous pcrs of this amplimer and check that the no template controls in earlier pcrs are negative
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