What is ideal DNA concentration?
DNA template concentration. Normally used concentration are 100-250 ng for mammalian genomic DNA and 20 ng for linearized plasmid DNA (circular plasmid DNA is slightly less efficiently amplified) per 50µl reaction.
What is the minimum amount of DNA needed for PCR?
Template DNA Nevertheless, the composition or complexity of the DNA contributes to optimal input amounts for PCR amplification. For example, 0.1–1 ng of plasmid DNA is sufficient, while 5–50 ng of gDNA may be required as a starting amount in a 50 µL PCR.
What is the optimal quantity of DNA for most commercial STR kits?
0.5 to 2.0 ng
Multiplex STR typing works best with a fairly narrow range of human DNA – typically 0.5 to 2.0 ng of input DNA works best with commercial STR kits.
How do you calculate DNA concentration in PCR?
DNA and RNA concentrations can be determined using their optical density measurements at 260 nm (OD260). The mass of purified nucleic acids in solution is calculated at 50 μg/ml of double stranded DNA or 40 μg/ml for either RNA or single stranded DNA at an OD260 =1.0.
What is a good DNA concentration ng UL?
for DNA sizes above 500bp, it is recommended the minimum concentration is 40ng/ul with a minimum volume of 15uL. for sizes below 500bp, 20ng/uL is sufficient.
What is a good 260 280 ratio for DNA?
The ratio of absorbance at 260 and 280 nm is used to assess DNA purity. A ratio of ∼1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower (≤1.6), it may indicate the presence of proteins, phenol, or other contaminants that absorb strongly at or near 280 nm.
How is DNA concentration calculated?
DNA concentration is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA.
How much DNA is needed for a STR analysis?
Introduction. Short tandem repeats (STRs) are short repeated sequences of DNA (2–6 bp) that account for approximately 3% of the human genome (Lander et al., 2001). The number of repeat units is highly variable among individuals, which offers a high power of discrimination when analyzed for identification purposes.
Why is 260 nm used to estimate DNA concentration?
Nucleic acids strongly absorb UV light with wavelengths of 260 nm due to the resonance structure of the purine and pyrimidine bases . The absorbance is converted into ng/μL of double stranded DNA (dsDNA) using the established conversion factor of 50 ng/μL for 1 optical density unit at 260 nm .
How does NanoDrop measure DNA concentration?
If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close the lid and click measure, be sure to record the concentration and purity. Note: Purity is measured under the 260/280 column (A good purity ranges from 1.80-2.00). Repeat for each sample.
How much DNA can I dilute to 50µl?
If it’s a plasmid, from 1pg to10ng in 50µL reaction is OK as Daniel said but if it’s genomic DNA you will have to consider hundreds of ng (up to 250ng per 50µL) has your target will be much more diluted in gDNA than in a plasmid. The ‘multiple bands’ can be associated with several factors.
What is the optimal amount of DNA for plasmid transfection?
The optimal amount of DNA varies depending on the characteristics of the transfected plasmid (e.g., promoter, size of plasmid, origin of replication), number of cells to be transfected, size of the culture dish, and the target cell line used. In many of the cell types tested, relatively small amounts of DNA are effectively taken up and expressed.
How much DNA is needed to kill a cell?
While with some cell lines 10–15 μg of DNA added to a 10-cm dish results in excessive cell death and very little uptake of DNA, other cell lines, especially primary cells, much higher concentrations of DNA is required.
What is the minimum amount of DNA required for 2415bp PCR?
Although 10ng of input DNA concentration is most commonly used, but for such size (2415bp) I would suggest to use higher concentrations of input DNA (30-50ng) in 20 µL reaction mixture. For long-range PCR between 10 and 30ng should suffice within a 50 microliter reaction.