Quantitative PCR Session 4: Plate Preparation and Data Analysis

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Quantitative PCR Session 4 Plate Preparation
and Data Analysis

• Presented by
• Robert O'Brien
• Training Specialist Forensic Biology

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Quantitative PCR

• Session 4- Plate preparation and Data analysis
• Importance of pipetting technique with
  Quantifiler
• Preparing standards
• Preparing reactions
• Avoiding and detecting contamination during qPCR
  set up
• Starting a run
• Analyzing data
• IPC
• Using quantifiler results

3
Importance of pipetting technique with
Quantifiler

• The standards in quantifiler are the basis by
  which all quantity determinations are made using
  the SDS software.
• All quantities of samples are calculated based on
  the curve that is made from the standards.
• Because of this great care must be taken when
  preparing the standards.
• Most problems associated with quantifiler runs
  and the subsequent data are due to problems with
  standards which are usually because of mistakes
  in pipetting.

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Importance of pipetting technique with
Quantifiler

• Tips for improving accuracy and precision when
  pipetting
• 1) Prewet the Pipette tip
• Draw in and expel sample at least 3 times before
  drawing in sample for delivery
• Purpose is to prevent evaporation that can occur
  in the tip. Prewetting the tip increases the
  humidity within the tip and therefore reduces the
  amount of sample evaporation

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Importance of pipetting technique with
Quantifiler

• 2) Work at room temperature
• Allow liquids and equipment to equilibrate to
  room temperature
• Volume of sample delivered by air displacement
  pipettes varies with air pressure, relative
  humidity and vapor pressure of the liquid, all of
  these are affected by temperature. Working at a
  single constant temperature minimizes this
  variation

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Importance of pipetting technique with
Quantifiler

• 3) Examine the tip before dispensing the sample
• Look at the outside of the tip to see if there is
  any liquid present.
• Liquid on the outside of the tip will increase
  the amount of liquid being dispensed. The outside
  of the tip can be wiped before delivery of tip
  contents but this must be done carefully because
  if the material being used to wipe the tip
  contacts the tip opening it can absorb the liquid
  inside of the tip

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Importance of pipetting technique with
Quantifiler

• 4) Use Standard Mode pipetting
• Choose standard mode pipetting rather than
  reverse mode pipetting
• Standard mode pipetting Push button to first
  stop of pipette then place pipette tip in liquid
  approximately 4mm below surface. Slowly release
  button to draw up liquid.
• To dispense slowly push button to first stop and
  then push all the way to second stop.
• Speed of drawing up and dispensing liquid should
  be the same

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Importance of pipetting technique with
Quantifiler

• Reverse mode pipetting Push button to second
  stop to draw up liquid and only push to first
  stop to dispense. (Reverse mode is used for
  liquids with a high viscosity)

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Importance of pipetting technique with
Quantifiler

• 5) Pause consistently after drawing up liquid.
• Pause with the tip in the liquid for one or two
  seconds after drawing up the sample. The amount
  of liquid in the tip bounces slightly when the
  plunger stops. Slow even plunger release and a
  consistent brief pause after drawing up the
  liquid minimizes errors resulting from the
  bouncing

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Importance of pipetting technique with
Quantifiler

• 6) Pull the Pipette straight out
• After drawing up the sample pull the pipette
  straight out.
• Do not allow the tip to touch the sides of the
  container.
• With very small volumes lt 50µl surface tension
  effects cause the sample volumes to vary if the
  exit angle varies. Touching the tip against the
  sides results in loss of sample.

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Importance of pipetting technique with
Quantifiler

• 7) Minimize handling the pipette tip
• Avoid handling the tip and set the pipette down
  between sample deliveries. Body heat transferred
  to equipment during handling disrupts temperature
  equilibrium. Already stated was that temperature
  can affect volume of sample delivered.
• In forensic science work, the pipette tip should
  never be handled.

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Importance of pipetting technique with
Quantifiler

• 8) Immerse the tip to the proper depth.
• Immerse the tip 2 to 5mm below the meniscus and
  well clear of the container walls and bottom when
  drawing up the sample.
• Inserting the tip too far into the liquid causes
  excess droplets of liquid to cling to outside of
  the tip. Pressing or resting the tip against the
  container walls or bottom restricts entry of
  sample

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Importance of pipetting technique with
Quantifiler

• 9) Use the correct pipette tip.
• Securely attach a tip designed for use with the
  pipette. Mismatching a tip and pipette or using
  poor quality tips can result in an inadequate
  seal between the pipette and the tip. Quality
  tips are flexible and have thin walls providing
  an airtight seal and more dependable delivery of
  the sample.

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Importance of pipetting technique with
Quantifiler

• 10) Use constant plunger pressure and speed.
• Depress and release the plunger smoothly with
  consistent pressure and speed for each sample.

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Preparing standards

• There are 8 standards that are run in duplicate
  on every plate. The highest concentration is
  50ng/µl and the lowest concentration is
  0.023ng/µl.
• The first standard is made with the DNA standard
  provided by Applied Biosystems that comes with
  the Quantifiler kit.
• To this standard is added TE buffer or a TE
  buffer glycogen mixture.
• (glycogen is added to the TE so the standards
  will last longer once made)

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Preparing standards

• From this initial standard and TE buffer mixture
  subsequent standards are made following a
  dilution series.

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Preparing standards

• The H standard in Human kits contains 14 to 16
  copies of a diploid single copy locus.
• The H standard in the Y kit contains 7 to 8
  copies of a haploid single copy locus.

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Preparing reactions

• Each well in the 96 well plate must have 23µl of
  PCR primer and reaction mix and 2µl of sample to
  give a total volume of 25µl.
• The primer/reaction mix is made up with 10.5 µl
  of quantifiler Human Primer (or Quantifiler Y
  primer depending on assay being conducted) and
  12.5 µl of quantifiler reaction mix per well.
• When calculating how much of each to use make
  sure to include samples, standards and controls
  and a few extra for pipetting error.

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Preparing reactions

• First step is preparation of Standards
• Label 8 tubes for the standard series
• Vortex quantifiler human standard
• Pulse spin
• Prepare serial dilutions using the quantities in
  the table previously shown
• Remember the standards are done in duplicate for
  each assay.
• During the preparation of standards great care
  must be taken when doing the dilutions. Change
  pipette tip when going from one standard to the
  next during dilutions.

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Preparing reactions

• Before making master mix swirl the reaction mix
  by hand. Do not vortex the reaction mix bottle.
• Vortex the primer mix and pulse spin.
• Make up the master mix, 10.5µl of primer mix and
  12.5µl of reaction mix per well.
• Briefly vortex the master mix and pulse spin
• Dispense 23µl of master mix into each well being
  used

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Preparing reactions

• To the appropriate wells add 2µl of standards
  that were made and to the other wells the samples
• Seal the plate with an optical adhesive cover,
  sealing all edges and between well spaces with
  sealing tool
• Remove any air bubbles that are in sample wells
  by centrifuging the plate. This is necessary
  because bubbles in plate can show up as small
  amounts of DNA when there is no DNA present.

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Avoiding and detecting contamination during qPCR
set up

• Avoiding contamination during Quantitation
• Even though the 2µl of sample being used for
  quantitation will not be carried to STR
  amplification it is still important to avoid
  contamination of one sample with another.
• If two samples are mixed during set up of the
  plate then the quantities will be wrong and the
  incorrect amounts of sample will be taken on to
  the amplification process.
• There can be cross contamination from one sample
  to another, contamination from an outside source
  from the person setting up the plate or from
  amplified DNA

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Avoiding and detecting contamination during qPCR
set up

• Avoiding contamination during Quantitation
• Ways to prevent sample to sample contamination
• 1 Clearly label extraction tubes and only have
  one tube open at a time when loading the plate.
• Arrange extraction tubes in the same order as
  they are going into the plate and move the
  extraction tubes after the sample is loaded to a
  different location to avoid the same sample being
  used twice.
• 3 Strip caps can be used to cover a row of wells
  on the plate after they are loaded. This prevents
  loading the same row of wells twice. It also
  prevents passing over loaded wells with a tip
  that contains DNA.

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Avoiding and detecting contamination during qPCR
set up

• Avoiding contamination during Quantitation
• Ways to prevent sample to sample contamination
• 4 When loading samples load them as close to the
  bottom of the well as possible and do not
  depress the pipette any further after it is
  pulled out of well.
• 5 Once the plate is loaded and sealed with
  optical adhesive cover do not tilt or turn the
  plate in such a way that would cause the samples
  to get onto the cover.

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Avoiding and detecting contamination during qPCR
set up

• Preventing contamination from an outside source
• Always wear PPE while loading plates. Especially
  face masks and gloves.
• Ensure the area where the plate is being set up
  in has been cleaned with bleach solution before
  plate set up.
• Have a separate room for plate set up and where
  the instrument is located. The instrument is
  amplifying DNA so it should be in a room
  dedicated for amplification. Set up should be in
  a room where there is not amplified DNA.

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Avoiding and detecting contamination during qPCR
set up

• Detecting contamination
• An optional blank can be loaded into the plate
  during plate set up.
• This is different from the extraction blank
  because this will specifically detect if there
  was any contamination introduced during plate set
  up or if the plate was contaminated before being
  used.
• The most common type of contamination problem
  with setting up plates is well to well
  contamination because unlike tubes it is
  difficult to cap each well after it is loaded.
• It is important to keep an eye on your results.
  If a sample that you expected results gives you a
  negative and the sample next to it gave extremely
  high results then it is possible that a well was
  double loaded and the next well was skipped.

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Avoiding and detecting contamination during qPCR
set up

• Detecting contamination
• Another indication of doubling of samples in a
  well is if a sample is run for both Total Human
  and Y and the Y gives a much higher result than
  the Total Human then it is possible that the Y
  well was double loaded.
• A lot of ways to detect contamination comes from
  thinking about results obtained from quantitation
  and determining if the results make sense. This
  will become easier with experience.

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Starting a run

• Instrument operation
• Turn on the instrument and allow it time to start
  up
• Open the AB Prism 7000 or 7500 SDS software,
• set up the plate document for the sample run.
  Usually there will be a template document that
  can be opened so only sample names will need to
  be filled in.
• Make sure the correct template is selected.
  Templates may vary based on the assays being
  performed (Total human only or Human and Y
  combination plate).

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Starting a run

• Once the sample names are filled in on the Plate
  document, then check the thermal cycler
  conditions.
• Ist step should read 95ºC for 10 minutes
• 2nd step 40 cycles at 95ºC
• 3rd step hold cycle for one minute at 60ºC
• Verify that the sample volume is set to 25µl and
  that the 9600 Emulation box is checked.
• Save the Plate document when you have finished

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Starting a run

• For 7000 place a compression pad on top of sealed
  plate, gray side down directly over the reaction
  wells.
• For the 7500 no compression pad is needed.
• Place the plate in the instrument and close the
  door and start the run.

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Analyzing the Data

• Once the run is complete the plate must be
  analyzed
• First verify all the analysis settings
• Threshold set at 0.20000
• Baseline start cycle 6
• Baseline end cycle 15
• If any of these are different they must be
  changed before analysis. If after plate is
  analyzed it is noticed these values are not
  correct then they can be changed and the plate
  reanalyzed.

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Analyzing the Data

• To view analysis settings before analyzing plate
  under Analysis go to Analysis settings the screen
  should be the same as this one.

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Analyzing the Data

• If the plate is already analyzed then check here
  for analysis settings

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Analyzing the Data

• To analyze plate go to Analysis and select
  analyze or click green arrow shown below

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Analyzing the Data

• After analysis of plate is complete the following
  should be reviewed
• The standard curve
• The amplification plot
• The report

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Analyzing the Data

• The standard curve
• The standard curve gives the following
  information
• The R² value of regression line which shows how
  well the line fits with values from standards
• The slope which gives an indication of efficiency
• The Y- intercept
• To select the standard curve under Results tab
  got to Standard curve verify slope for human is
  between
• -2.9 to -3.3 and for Y -3.0 to -3.6
• R² should be gt 0.98

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Analyzing the Data
38
Analyzing the Data

• The values for the R² , and slope should be in
  the ranges shown for the data to be used.
• If these values are not within range then there
  may have been a problem when setting up the
  standards that made the standard curve. This
  reinforces how careful one must be when making
  standards since if these values are not in range
  the plate may not be able to be used and the
  procedure may have to be repeated.

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Analyzing the Data

• Only with the standards for a Y plate can an
  adjustment be made.
• The STD 8 or H for the Y kit only has 7 to 8
  copies per 2µl of the haploid target locus.
• Because of stochastic effects when using such a
  low amount of DNA the CT values for this
  concentration of DNA are more variable. This
  variability can throw off the line of best fit
  and this in turn will throw off R² value.
• So the STD 8 or H for the Y kit can be dropped by
  omitting the wells and reanalyzing.

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Analyzing the Data

• To omit well select the well then go to Well
  inspector and click on omit well

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Analyzing the Data

• After well is omitted under the Plate tab the
  well should be crossed out. Notice that the arrow
  is green indicating reanalysis is necessary.

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Analyzing the Data

• Troubleshooting Amplification Plot
• R² value lt 0.98
• Quantity values entered for quantitation
  standards in well inspector are incorrect
• Serial dilutions were not made properly
• Standards were not loaded correctly
• Reaction for one of the standards failed
• Slope not in range
• Assay not set up correctly
• Software not set up correctly
• Reagents may be expired or wrong ones used
• Instrument may malfunction

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Analyzing the Data

• Y intercept
• There is no fixed value for the Y intercept
• The Y intercept is the expected CT value for a
  sample that has a quantity of 1ng/µl
• The Y intercept may shift from one kit lot number
  to the next. The Y intercept from all runs
  performed with a certain kit lot number should be
  within an acceptable range of the intercept value
  when the kit was quality checked. The range is
  lab dependant.

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Analyzing the Data

• After the plate is analyzed then the
  amplification plot is checked to see what samples
  gave results and which were negative for DNA.
• The amplification plot is a plot of ?Rn vs Cycle
  number.
• This screen shows the corresponding wells for the
  samples. Each individual sample can be checked to
  see if the sample crossed the Threshold or not.
  Samples not crossing the threshold are negative
  no DNA present.
• Also the IPC can be checked for all of the
  samples.
• All IPCs should cross unless inhibition occurred.

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Analyzing the Data
46
Analyzing the Data

• The individual wells can be selected one at a
  time
• Also putting the cursor on a plot will show which
  sample it corresponds too.
• Under Detector there is a drop down menu to
  select.
• ALL, IPC, Quantifiler Human, or Total Y

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Analyzing the Data
48
Analyzing the Data

• To see either the Quantifiler Human or IPC by
  itself then go to Detector drop down box and
  select what plot you would like to view

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Analyzing the Data

• The report
• The report will show the quantities calculated
  for the samples.
• The IPC well has no value under quantity because
  no quantity calculations are made for IPC,
• If more than one sample has the same name then
  they are grouped together by the software and the
  Mean value and Standard deviation for the samples
  are calculated.
• During practice plates duplicating samples is a
  good way to test precision of pipetting technique.

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Analyzing the Data
51
IPC

• IPC Internal PCR control
• IPC is included as part of each reaction well. It
  undergoes the same PCR reaction as the sample but
  because its target sequence does not exist in
  nature it does not compete with the primers and
  probes that target human DNA.
• However because IPC is undergoing PCR along with
  the sample anything that negatively affects PCR
  of sample will also affect PCR of IPC.

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IPC

• Since the IPC is affected by problems with PCR
  that affect the sample it can be used as a
  quality control check.
• IPC can detect the following
• Presence of inhibitors
• Problems with Assay set up
• Chemistry or Instrument failure

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IPC

• If the IPC crosses the threshold and the sample
  does not then the sample is considered a true
  negative.

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IPC

• The CT range for the IPC VIC dye should be
  between 20 to 30,

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IPC

• If both the Human specific target and the IPC
  fail to amplify it is not possible to determine
  if the negative is due to no DNA present to
  amplify or if it is due to something affecting
  the PCR reaction like inhibition.
• If inhibition is suspected then the sample can be
  cleaned.
• Cleaning can be done by concentrating the sample.
  Some inhibitors will pass through the filters
  during the washes.
• If the sample was already concentrated then it
  can be diluted. This action can reduce the
  concentration of the inhibitor and in turn reduce
  its effect on the PCR reaction.

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IPC

• If neither of these work then the sample can be
  re-extracted using a cleaner extraction method.
  Some extraction techniques like organic
  extraction produces a cleaner end product.
  Inhibitors are usually removed during organic
  extraction.
• If this is not possible then a new sample may be
  taken from the evidence that may be free of dyes
  or dirt or other materials that could have caused
  the inhibition.

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IPC

• If the IPC does not cross the threshold and the
  sample has a low CT value and a high ?Rn that
  means there is a lot of target DNA present. In
  this case the DNA may have out competed the IPC
  for certain components necessary for
  amplification.

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IPC

• Earlier it was said that the IPC and target DNA
  have different primers so they did not compete
  with each other for those. However there can be
  competition for polymerase and other components
  like Mg needed for PCR.
• If the sample has a high CT value and a low ?Rn
  and the IPC did not amplify it could mean there
  was partial inhibition. Depending on how low or
  high the quantity is the sample may still amplify
  and give a result during STR amplification or the
  sample may have to be cleaned up before
  continuing with the DNA analysis process.

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Using Quantifiler results

• Should a sample that gives a negative result be
  taken on for further DNA analysis?
• Some laboratories choose not too carry the sample
  any further in an attempt to conserve money and
  time. During quantifiler the sample is put
  through 40 cycles of PCR. The PCR that occurs in
  the next stage of DNA analysis only puts the
  sample through 28 cycles. Theoretically if a
  sample does not amplify in 40 cycles it will not
  amplify in 28 cycles.
• As a result a true negative quantifiler result
  will not be expected to generate a profile. The
  practice of dropping a sample after quantitation
  is laboratory dependant.

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Using Quantifiler results

• If after quantitation there is DNA in your sample
  then the extract must be taken on to STR
  amplification.
• The final value from quantitation will be in the
  units
• ng/µl
• This simply means that for every 1 µl of extract
  there is a certain amount of DNA.
• STR amplification kits require a certain amount
  of input DNA to give optimum results. Meaning a
  full profile with good peak heights.
• Usually targeting an input of 1ng of DNA for STR
  amplification will produce a good profile.

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Using Quantifiler results

• If final quantity is 1ng/µl then by simply adding
  1µl one will have the necessary amount of target
  DNA.
• What if value is 4.25 ng/µl.
• For every 1µl there is 4.25 ng of DNA. So 1µl of
  extract will be greater than optimum input level
  for the amplification kit. This sample needs to
  be diluted before it can be amplified.
• Calculation
• 4.25ng 1µl
• 1ng 1 / 4.25 0.235µl

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Using Quantifiler results

• Calculation
• 0.235 µl of a sample is too little to be added.
  Pipetting error is common when using such low
  quantities of sample. By diluting the sample,
  more of diluted sample can be added and pipetting
  errors can be reduced.
• Dilution by a factor of 10
• 1 µl of extract to 9µl of TE buffer.
• After dilution by a factor of 10 simply multiply
  volume by 10
• 0.235µl x 10 2.35 µl
• So for a quantity of 4.25ng / µl. It should be
  diluted by a factor of 10 and 2.35 µl of diluted
  sample be added to amplification tube.

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Using Quantifiler results

• At end of quantitation the quantity of DNA is
  0.365ng/µl.
• So 1µl of sample will not give 1ng of DNA.
• To calculate how much µl will be needed to give
  1ng of DNA.
• 0.365 ng 1µl
• 1 ng 1/ 0.365 2.73 µl
• So you will need to add 2.73 µl of extract to get
  1 ng of DNA for amplification.

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Using Quantifiler results

• If you are using a 25 µl reaction for the STR
  amplification then the amount of sample being
  added is limited.
• If the quantity of DNA is too low then it will
  require more than the amount of DNA that can be
  added to the reaction.
• In these cases it is advisable to concentrate DNA
  and quantitate again before going on to
  amplification.

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Using Quantifiler results

• At the end of this process samples should be
  ready for STR amplification.
• As long as the quantifiler results show there is
  enough DNA that the amplification kits require to
  generate a full profile then the analyst should
  be confident they will be able generate a full
  profile at end of STR analysis.

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Using Quantifiler results

• One important limitation to consider is if there
  is a mixture of DNA in the sample the quantity
  generated is how much total DNA is present.
• The resulting profiles may be two or more
  incomplete profiles or one full profile and one
  incomplete profile.
• Total Human quantitation cannot tell if the final
  value is from more than one person.