gDNA contamination in cDNA library with cDNA creator smart kit - (Jan/07/2007 )
Has anybody experienced contamination with genomic DNA in a cDNA library using the Clontech "creator smart cDNA library construction kit"?
In principle, no DNA would be amplified, not cut with the enzyme and not ligated... However, some clones do seem to be gDNA, based on GenBank sequences.
I know there are errors on data banks. Is there a way to diagnose gDNA based on the sequence and not on data bank?
You could try translating the sequence and see if a stop codon appears. If introns are long enough, this usually happens. For shorter introns, you could check to see if there's a frame shift that also will eventually lead to a stop codon (ie. intron length is not a multiple of 3). For short introns that are a multiple of 3 in length, you could run the sequence through a programme that predicts intron/exon junctions, although false positives and negatives are common with these programmes. Do you get multiple products with a PCR?