In my recent experiment, a plasmid containing the full-length insert of the DHBV genome is needed to prepare the standard curve for fluorescence quantitative PCR assay for detection of DHBV-DNA. But it is not that easy for me to get this plasmid from ATCC. So I plan to construct this plasmid by myself via inserting the product of the amplification of nucleic acid extraction from liver tissue using a pair of specific primers for the full-length insert of the DHBV genome. If the plasmid is confirmed to be constructed as expectation by enzyme digestion and sequence reaction. Can I declare that I have constructed a plasmid containing the full-length insert of the DHBV genome? Do I need any further strategies to confirm this plasmid? Or I should try to get a standard plasmid from other people rather than constructing it by myself?
Hi, If want to find out the construct is correct or not, you might need to sequence it.
By the way, if you just want to construct a standard curve, why do not only clone PCR region? I think it is enough for a standard curve. If you will need full length DHBV for experiment, full-length DHBV construct will be necessary.
Good Luck
For qPCR you only need to clone your sequence target plus 100 bp up- and down-stream to ensure a proper qPCR amplification reaction. The full sequence is not necessary to perform your qPCR detection analysis.
Thank you for your answser. Since I plan to detect the level of cccDNA and rcDNA of DHBV, so I guess a plasmid containing the full-length insert of the DHBV genome would be necessary.
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