I want to isolate the promoter of my gene for promoter analysis. so i need to isolate the full length sequence of promoter. So can anyone tell me the most commonly used method for isolation of promoter.
Thank you.
Hi Harshraj,
If you dont know gemomic fragment of your gene, By primer walk through inverse PCR your will get upstream region of your gene. This sequence you can use as input for promoter prediction through different software. Then after promoter prediction you have to validate the promoter activity through cloning and expression of gene under this promoter.
Hi Harshraj,
Do you want to know the procedure of finding the promoter of a gene or cloning!
Swati
I want to know procedure of finding the sequence of promoter of a gene.
Here is a link where you can find the promoter region and transcription start site of a gene. You will have to input around 1-1.5 kb region upstream of your gene of interest. TSSP is software you should use for plant promoters. You can find more promoter finding softwares online aw well.
Best,
Swati
http://linux1.softberry.com/berry.phtml?topic=plantprom&group=data&subgroup=plantprom
Hi Swati. Thank you for information.
Actually I know the sequence of my gene but i dont know the sequence of promoter so i want to isolate the sequence of promoter. so what are all the methods/techniques available for isolation of promoter in plants?
You need the upstream region of gene, which you can get if you blast it against the assembly of your organism.
Actually i did nblast, but in blast results i cant see any nucleotide sequence before ATG
Do not do nblast. I would suggest Blasting the assembly....you can find the assembly in NCBi.
Dear Hunsraj
Can you tell me that whether the gemone sequence of the crop is available in which you cloned the gene I mean your Gene of Interest. If yes, then simply you can map your gene in genome browse of the genomic database of the crop and based on the orientation of your gene you can select the upstream (appox. 2.0 kb) sequences from primer designing. Take these primers for amplifying the promoter region using genomic DNA of your crop and validate through sequencing.
If not, then you can use TAIL-PCR, inverse PCR or chromosome walking as usual and most commen method of promoter isolation. You can get the protocols about these methos easily at website. If you you want some more details then let me know.
Good luck
Regards
Chet Ram
Thank you Ram For answer. Genome sequence of crop I am working is not available so I am doing TAIL PCR. I got few online literature regarding TAIL PCR. Do you have any lab protocol of TAIL PCR? please share if you have
Regards
Harshraj
Hi Harshraj,
If you dont know gemomic fragment of your gene, By primer walk through inverse PCR your will get upstream region of your gene. This sequence you can use as input for promoter prediction through different software. Then after promoter prediction you have to validate the promoter activity through cloning and expression of gene under this promoter.
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