Hi Arabidopsis researchers,
I recently want to screen out the homozygous cbf2 gene Salk T-DNA
insertional mutant lines by PCR according to Salk Institute protocol. The
Salk lines we ordered from ABRC are SALK_009689c (LOCN 1000-Promotor) and
Salk_025195(LOCN 300-UTR5). However I failed to get true knock-out mutants
after running RT-PCR, although PCR results using LBb1/RP and LP+RP showed
that I got the putative homozygous lines. This can be partly explained by
the fact that T-DNA insertion in the promoter region may not influence the
transcription of CBF2 in Salk_009689, but I expect T-DNA insertion near ATG
start codon in Salk_025195 should block the transcription of CBF2.
One paper in PNAS(2004): CBF2/DREB1C is a negative regulator of CBF1/DREB1B
and
CBF3/DREB1A expression and plays a central role in stress tolerance in
Arabidopsis. In this paper, the authors reported a Salk line HM mutant-no
detectable CBF2 transcript by Northern blotting. I then asked the
corresponding author a few months ago but no reply at all. So I have no way
to know which Salk line they used.
Could anyone working with CBF2 mutant reply?
Thanks,
Luoping
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