Currently i am working with ABRC mutant lines of arabidopsis and during PCR for T-DNA genotyping my results are not reproduceable. Like i got bands in LB and RP and there is no band of same primer set in WT but after harvesting seeds from homozygous plant when i repeat the same method with leaves of new plants i got nothing in PCR even there is no band with LP-RP primer set in WT what could be the reason of this?
these are some gel images shared
There might be a problem with either your PCR or the genomic DNA extraction. Do you still have the old genomic DNA samples? in this case you can repeat the new PCR and include one positive control from the previous extraction for each PCR. If that comes in positive, it's likely the new extraction that has problems, maybe low concentration or purity. You can check concentration, and integrity of the DNA prep (spectrophotometer OD; and run on gel). Usually with a not so clean extraction, 100-200 ng as template per reaction works).
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