Hi everybody,
I now encounter a overwhelming problem about genotyping. after first round genotyping, I selected the positive seedlings,i.e., with FP+RP, no wide type DNA was amplified, with LB+RP, a T-DNA insertion DNA was amplified. OK now question comes. after I select this positive mutant, and got next generation seeds from this plant, and genotyped once again. There is a weak band same as wild type size in mutant. I even changed new primers, and still weak bands. I still selected positive plants without this weak bands, and got seeds from them, and genotyped once again, this weak bands appear gain. I don't know how to deal with. I was crazy. Btw, this SALK line has been reported by others as loss of function, and no band can be amplified in this mutant when genotyping.
any suggestions will be welcomed!
1. Assuming that you already obtained a homozygous mutant line
2. Can you re-design another pair of FP + RP to see whether any (faint) wild-type band show up in the mutant?
When you did PCR, did you use 'wild-type' DNA as a control? Some very few amount of them might be carried-over to the PCR tubes for 'mutants' through pipetting and aerosol. You may want to try the use of tips with filter (filtered tips, see attachment) to see whether the 'noise' band can be eliminated.
By the way, is this faint 'wild-type' band in your no-DNA negative control?
Can you post your picture for us?
Did you blast both primer pairs? Is there is a close homologue of the gene with T-DNA insertion and that's what you're picking up?
The easiest may be to check contamination and you already did. I had the same crazy problem and I managed to publish why it can happen: Rodríguez M. C., Wawrzyńska A, Sirko A. Intronic T-DNA insertion in Arabidopsis NBR1 conditionally affects wild-type transcript level. Plant Signaling & Behavior 2014; 9:e34375.
Briefly, the T-DNA insertion in the homozygous mutant I was interested in was in an intron instead of in an exon (as claimed on TAIR and by Zhou et al. 2013); I mapped ethe exact location of the T-DNA insert. This caused that under certain conditions the T-DNA in the intron was retained (and the plants behaved geneticall as mutant, although I couldn't see any phenotype from these lines), while under other conditions the T-DNA was spliced together with the intron and the plants were genetically WT.
I had other similar experiences with T-DNA and I tried silening by antisense and iRNA but I got overexpressions or a maximum of 75 % KD (not a complete KO). You can screen for other mutants or try CRISPR-Cas9.
By the way, Zhou et al. used the same pictures for their paper of 2013 and 2014 for different lines and different phenotypes (compare e.g. their Figures 5B and 1B for 2013 and 2014, respectively, and find the identical plants).
Thanks a lot, Milagros Collados Rodríguez! very useful!
The mutant I have is also a intron T-DNA insertion. Actually, at least in two labs, they used the same mutant (same salk line) and didn't find any transcripts in this mutant. But when I used the same mutant from NASC, sometimes I found weak transcripts in the offspring of this mutant. I don't know why. Maybe contamination like what Yuan-Yeu Yau said, because of pipetting and aerosol. Because I re-redesign the third primers and did genotyping using the same genomic DNAs which I kept them from the second round genotyping(in the second round genotyping, I found weak transcripts, and I marked these DNA) . I found no transcript in this mutant this time. Btw, in the double mutant originated from this single mutant, I never find any transcript for this gene based on genomic genotyping. Therefore, I think, to large extent, it is because of pipetting and aerosol. Do you think so?
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