Back ground: I decide to transfer Gene related to Saccharum officinarum into the other plant and then express.
Then I just need ORF .I should design the primer for the first step after extracting RNA
I know this information:
AJ293346.1
gene 1..3067
gene="PEPC"
CDS 1..2886
The questions are:
1. Can I design only the pair primer which start from the first and end of ORF and make the specific sequence ( 2886 bp) or regarding to the length (2886 bp) I have to divide the sequence in three locations and then design primers at least with three ones . With considering which I finally have to transfer and express this mentioned length, is it right make 3 steps for my work with three primers or I have to make just one pair primer for this mentioned length.
2. With considering that gene and CDs start in the same location as I mentioned in back ground. If I start exactly from ATG in the start of the CDs, I cannot design a proper primer! The question is it:
1. Start of primer with ATG does not make problem?
2. If I decide to find around 50 mRNA nucleotide before the ATG of the ORF, but it is not mentioned in Gene sequence, how can I find by the whole genome sequence of sccharum officinarum. I know that the whole genome of saccharum officinarum has identified( Asano T, 2004), but I do not know how I look for 50 nucleotides before the ORF of the gene of interest.
Hi Dr. Jafarpour
As I figour out, you need to 3 different step for your expression process. I think I you use new synthetic plasmids which have 2 different ORFs, you can bypass your steps.
On the other hand, for confirming your cloning or subcloning, you can use T7 primer (universal primer) and only compare the size of your supposed gene. through this way you do not need to designing primer for some steps, too.
to be success
Bye
M.Sadraeian
Pharmaceutical Biotechnology Faculty
0098.9171038918
Thanks , Mohamad I tought you misunderesatand!but thank youfor responding my question.
Hi Jafarpour, the 2.8Kb gene is not much lengthy and you can amplify using a pair of primers bt you have to optimize PCR conditions. if you dont get success then you need to think abt alternatives i.e. primer designing between the sequence and amplifying the products and then ligating them all.
2. Primer start with ATG is not problem if it will not disturb the sequence downstream to ATG. afterall you have to mantian the downstream seq as well. the imp point to consider is cloning steps and restriction analysis you will go after amplification.
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