What is transgene copy number?
Transgene copy number is defined as the number of exogenous DNA insert(s) in the genome. For example, if the exogenous DNA fragment inserts only once at a single locus of the genome, it is a single copy transgenic event. The copy number is closely relevant to another concept, zygosity.
How do I find my transgene copy number?
The copy number can be determined by comparing the density of 3.3 kb band with3. 5, 4.5 and 5.6 kb single copy bands. #5, 6, 13 and 16 inherited all the copies of the transgene.
Which technique is used to know the copy number of transgene in transgenic plant?
Southern blot analysis, a classic molecular biology method, is traditionally used to determine transgene copy numbers.
How is gene copy number determined?
To measure DNA copy number, the amplicon should be located either within an exon or intron with sequences unique to that gene. A control gene with two copies should also be included. A master mix containing all of the components is prepared and distributed in 96 or 384-well plate.
How is transgene insertion site determined?
Classical methods to determine transgene insertion sites have utilized chromosome walking. A number of PCR-based methods are available for chromosome walking, such as inverse PCR (4), ligation-mediated PCR (5,6), and specific-primer PCR (7,8), which identify transgene flanking sequences.
How much genomic DNA do you need for qPCR?
For digestion, we recommend using 0.5-4ug (0.125-1ug per digestion) of genomic DNA for EpiTect Methyl qPCR Arrays and 0.25-0.5ug (0.0625-0.125ug per digestion) for single qPCR assays. However, you should not use less than 0.25ug (0.0625ug per digestion) of genomic DNA for digestion.
How many copies of a gene are there?
Every person has two copies of each gene, one inherited from each parent. Most genes are the same in all people, but a small number of genes (less than 1 percent of the total) are slightly different between people.
What is transgene insertion?
Transgenic technique1, whereby an exogenous gene is inserted into the mouse genome by direct injection of DNA into the pronuclei of a zygote, has enabled thousands of new transgenic lines to be created2. However the technique is not without limitations.
What is an endogenous gene?
Genetic normalization is performed by using endogenous reference genes. An endogenous reference gene commonly called “housekeeping gene” (HKG) is any gene that is stably expressed under all developmental and experimental conditions such as genes associated with metabolism.
How many copies can qPCR detect?
All Answers (7) Dear Cátia, most well optimized qPCR can have a limit of detection as low as 1 to 10 copies per reaction in optimal conditions, although the actual limit may be higher depending on each sample because of the presence of inhibitors, the quality of the DNA / RNA after each extraction procedure etc…
What is the optimal primer concentration for qPCR?
When designing primers, the amplicon length should be approximately 80–250 bp. A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a titration of primer concentrations between 100 and 500 nM.