I am willing to isolate bZIP28 transcription factor gene from arabidopsis, but having problem in extracting the gene.
Depending on what you want to use it for. You can start by looking for its sequence. Design primers. Isolating mRNA/total RNA, synthesize the cDNA and then amplify it from there. Or you can amplify it from the genomic DNA. If you dont have its sequence from Arabidopsis check where it has been cloned before in another organism and see if you can design degenerate primers.
I need the complete gene and have primers for it F primer from start codon ATG and R primers to end with restriction site and I also have cDNA.
When I am checking the expression level of bZIP28 with different pair of primers I am getting the expression, but when I am extracting the complete gene For CLONING, Its getting hard to extract.
thanks for Reply, but I need the gene for Cloning.
I already confirmed it with ORF gene primers.
My gene size is around 2200bp
I presume that you are isolating the gene from mRNA (by RT-PCR) and not from genomic DNA........
May be you can try using long range RT enzymes like superscript RT-enzymes and also long range taq polymerase capable of amplifying longer lengths.......
Alternatively the primers that you are using might be having some some problems eg..like formation of primer dimer etc.., you can try out with some alternate primer sequences if its possible.
Already the complete cds is available in NCBI..., as well as complete genome sequences of the Arabidopsis is available.......
http://www.ncbi.nlm.nih.gov/gene/820249
http://www.ncbi.nlm.nih.gov/nuccore/?term=bZIP28%20arabidopsis
http://www.ncbi.nlm.nih.gov/nuccore/KC412252.1
http://www.ncbi.nlm.nih.gov/nuccore/NM_111917.4
I do hope the above links will be useful
Dear Kaushal
you mean that for cDNA synthesis i should use higher concentration of RNA about 8.5ug and while doing cDNA synthesis I am adding oligodt in reaction mixture.
and sorry but I don't get your last point about Maximum RTase reaction, could you please elaborate that.
Thanks Regards,
Pratima
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