Hi. I want to synthesize this blaTEM gene. This is the FASTA sequence, but I don't know which part is related to coding sequence, Can anyone help me?
(my major isn't related to microbiology or genetic, so I don't know the exact procedure of genetic basics,)
and one more thing, after synthesis how can I know how many copy numbers are there in my sample to generate qPCR standard curve based on that?
The coding sequence start from the first "ATG" right after bacterial promoter sequence and end up at stop codon "TAA".
From 101 to 961.
You need to talk with your advisor BEFORE you set up any qPCR reactions. You need to know the concentration of your standard to know how many copies per unit volume. Most folks clone the region of interest into a plasmid and use the digested plasmid DNA to make their standard curve.
Read the MIQE guidelines for qPCR experiments & figure out what sort of analysis you are planning. You need to know exactly how you will be analyzing your data before you collect any.
The type of question(s) you are asking will help guide how you set up your qPCR reactions.
How many genes of interest? Are you using relative or absolute expression? How many biological replicates? How many technical replicates? etc.
Good luck!
Mahsa Maleki, the gene copy number can be arbitrarily determined by performing the nucleotide blast against the organism in which you want to determine the CNV. This will give you the rough idea on copy number, based on this you can choose other genes a control (single copy, multicopy).
To determine the CNV through PCR, you can clone the gene of interest and prepare a standard curve post serial diluting the plasmid DNA. Using a pure DNA (from organism of interest), extra plot the ct value obtained in the standard curve. This will give you the log copes of a gene.
First check for starting codon and stop codon, and continue the experiment.
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