33 Matching Annotations
  1. Last 7 days
  2. May 2024
    1. Do not exceed 5,000 copies of target/μl of the final ddPCR reaction mix

      1e5 targets per 20 ul reaction well. Sounds a little high no for 20,000 droplets, I would estimate a total of 2,000 copies to keep the avg 0.1 copies per droplet?

    2. Restriction Digestion

      See notes in this section for restriction digestion tips

    1. DNA fragmentation by restriction digestion prior to dropletgeneration enables optimal accuracy by separating tandemgene copies, reducing sample viscosity, and improving templateaccessibility for input samples >66 ng per well

      NEB says

      Digestion is recommended whenever DNA input is greater than 75 ng Source

      NEB says that biorad recommends these enzymes: AluI, CviQI, HaeIII, HindIII-HF, MseI

      More guidelines

      • Assemble ddPCR reactions at room temperature, this allows the restriction enzyme to digest the gDNA during the reaction set-up period
      • Prepare reaction mixes as you would for a standard ddPCR reaction. Add 0.5–1 μL of each restriction enzyme (5–20 units, depending on enzyme concentration) to the reaction mixture
      • After set-up, simply continue droplet generation as normal
      • Restriction enzyme will be inactivated during first PCR denaturation step
  3. Feb 2023
  4. Nov 2022
    1. For those shorter than 25 bases, a standard (dual labeled) BHQ™ Probe may be perfectly satisfactory.

      Nova might be better to have for 1-step ddPCR applications where the BHQ quenchers are unstable due to reduction of the N=N azo bond by the DTT additive

  5. Jan 2022
    1. process of dPCR is, for the most part, the same as that of qPCR; the key difference is that the nucleic acid sample is diluted and separated into numerous individual partitions that are independently amplified

      qPCR vs dPCR

    2. The copy number of nucleic acids is then calculated using Poisson statistics on the number of positive versus negative partitions22.

      Poisson for copy number in PCR

  6. Apr 2021
    1. the concentration is calculated based on the fraction of droplets that is empty (that is, the fraction that does not contain any target DNA).
  7. Mar 2021
  8. Dec 2020
    1. We recommend the following changes to the default settings when designing ddPCR assays:

      Primer3 : designing primers and probes for ddPCR

      In the General Settings window, change “Concentration of divalent cations” to 3.8, “Concentration of dNTPs” to 0.8, and “Mispriming/Repeat Library” to the correct organism ■In the Advanced Settings window, change both the “Table of thermodynamic parameters” and “Salt correction formula” to SantaLucia 1998 ■In the Internal Oligo window, we recommend setting 15 for the minimum number of bases for the oligo. We recommend 64°C as the minimum Tm for the probe, 65°C as the optimal Tm for the probe, and 70°C as the maximum Tm for the probe. These parameters can be relaxed to allow for smaller/larger oligos, which may be necessary for high GC or low GC targets. Oligo size should be no smaller than 13 and no larger than 30 nucleotides

      Note: After you have made the desired changes in Primer3Plus, select Save Settings under General Settings and save these parameters in a file. To apply these settings in the future, upload them by selecting Browse in the General Settings tab, find this file, and click Activate Settings.

    2. Strive for a Tm between 50 and 65°C. One way to calculate Tm values is by using the nearest-neighbor method. Use the Tm calculator at http://www.basic.northwestern.edu/biotools/oligocalc.html, with values of 50 mM for salt concentration and 300 nM for oligonucleotide concentration
  9. Nov 2020
    1. To detect foreign DNA in 5 ml of lake water, 15 ml of lake water must be screened.

      Why is this multiplied by 3? Related to statistical error of subsampling - rule of three)

    2. average of 5 copies/droplet (the upper end of the recommended loading range), we expect to see about 134 empty droplets in a total of 20,000 droplets
    3. At the extreme ends of the concentration range (for example, fewer than ten copies of target in a well or more than 120,000 copies of target in a well), fewer droplets in a well will lead to slightly larger error bars.
  10. Oct 2020
    1. Repeated freezing and thawing of the supermix is not recommended. DTT should be aliquoted to multiple tubes and stored at –20°C to minimize freezing and thawing
  11. Sep 2020
    1. LOD was defined as <x>bi + ksbi, where <x>bi equals the mean of the no-template controls, sbi is s.d. of no-template controls and k = 2.479 (99% confidence interval)


  12. Aug 2020
  13. Jul 2020
    1. There is a simple mathematical relationship between the fraction of droplets that are unoccupied (black bar) and the concentration of target molecules.
    2. Some droplets are lost in transfer steps and others are eliminated by the stringent metrics applied by QuantaSoft Software as the droplets pass through the Droplet Reader, resulting ultimately in data from 12,000–16,000 droplets being used in subsequent concentration calculations
    1. Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device.
    2. An estimate of par-titioning error can be found based on the analysis of Dube et al.,39 which models the partitioning as a binomial process.
    3. Digital assays have two sources of error: subsampling error and partitioning error
    4. Partitioning the sample into small containers results in a statistical distribu-tion of targets
    5. because it measures indi-vidual molecules rather than an ensemble concentration
    6. purifying the target of interest from interfering compounds.
    7. Subsampling error sets the lower detection limit at low concentrations and is independent of the instrument, while partitioning error domi-nates at high concentrations and may depend on the sampling and partitioning instrument
    1. The average number of droplets read for each ddPCR was 13,825 with a standard deviation of 1,892 droplets
    2. NTCs showed a low-level background signal of approximately three positive droplets per NTC assay, which could possibly be attributed to low-level template contamination during the preparation of the reaction mixture
    3. simple calibration procedure generated an instrument-specific color compensation matrix that was subsequently stored on the droplet reader and automatically applied to data to eliminate cross talk between FAM and VIC labeled probes.
    1. Poisson distribution can be used in place of other standard curves to determine template concentration, which means that data can be reliably obtained from the same samples across multiple instruments and laboratories.