General DNA Quantification
For each plate:
- 96 well black polystyrene plate
- 300uL tips- Rainin
- Multi-channel pipette-Rainin 8-channel
- Sterile reservoir
- non-glass container (ex. 50mL conical)
- lambda DNA standard 100ng/uL (in kit)
- PicoGreen reagent (in kit)
- 20x TE buffer (in kit)
- HPLC water
PicoGreen Prep
1. Per Well Volume 150 uL/well
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- a)Use 110 sample number per plate
- i) 110 sample x 150uL =16.5mL
- a)Use 110 sample number per plate
2. Using 20x TE buffer make 1x solution
3. Add 5uL of PicoGreen reagent for every 1mL of buffer
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- a) 16.5mL of TE x 5uL PicoReagent = 82.5uL PicoReagent
- i) PicoGreen is light sensitive and will undergo photo degradation
- ii)PicoGreen contains DMSO
- a) 16.5mL of TE x 5uL PicoReagent = 82.5uL PicoReagent
Quantification Assay
1. Pipette (multi-channel) amplicon into appropriate well
2. Add 150 ul of PicoAssay to samples, MIX WELL by pipetting
3. Concentration standard
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- a)2uL- lambda DNA in the top well of column
- b)Add 150uL of assay to the entire column
- c)Add an additional 150uL to the top well (3a)
- i) Serial dilutions down the column with 150uL transfer
- a) The last well be 0 point
- i) Serial dilutions down the column with 150uL transfer
Measure Fluorescence
1. Turn on BioTek Synergy HT plate reader
2. Open Gen 5 program from desktop
3. Select “Experiment”
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- a)PicoGreenEx.prt
4. Select “Read Plate” from icon in toolbar
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- a)Name file- mm/dd/yyyy_projectname_plate#?
5. Place pate on carrier, press “OK”
6. Save as an Excel file
Modified Protocol
For each plate:
- 96 well black polystyrene plate
- Single channel pipettors and tips
- 8-Channel pipette and tips
- Sterile reagent reservoir
- Non-glass container to dilute and mix PicoGreen reagent
- PicoGreen stock reagent (component A–Invitrogen P99496) keep in the freezer
- 20X TE (component B) keep in the fridge
- Lambda DNA standard (component C) keep in the fridge
- HPLC water
1. PicoGreen Dilution (for 96 quantification + volumes loss during pipetting (110 total aliqouts of diluted PicoGreen reagent)
2. Dilute 20X TE buffer in HPLC water to make 1x solution
- aliquot 15.7mL HPLC water into clean non glass container (e.g. conical tube)
- aliquot 825 uL 20x TE into HPLC water
- total volume ~16.5mL (150uL diluted PicoGreen per sample x 110 aliqots= 16.5mL)
3. Aliquot 82.5uL PicoGreen stock reagent into the 1X TE (thaw in the dark and not in the fridge–hold in hand/pocket)
- PicoGreen is light sensitive and will undergo photo degradation
- PicoGreen contains DMSO
4. Quantification
5. Pipette 2uL of amplicon per sample into appropriate well on the black plate
6. Pour diluted PicoGreen reagent into the reagent basin
7. Add 150uL of diluted PicoGreen reagent to each sample, mix by pipetting, conver to protect from light (can seal with aluminum foil)
8. Prepare 7 point standard with blank by serial dilution
| Well | Contents | Action |
|---|---|---|
| A1 | 2uL lambda DNA standard + 300uL diluted PicoGreen reagent | Mix and transfer 150uL from A1 to B1 |
| B1 | 150uL diluted PicoGreen reagent | Mix and transfer 150uL from B1 to C1 |
| C1 | 150uL diluted PicoGreen reagent | Mix and transfer 150uL from C1 to D1 |
| D1 | 150uL diluted PicoGreen reagent | Mix and transfer 150uL from D1 to E1 |
| E1 | 150uL diluted PicoGreen reagent | Mix and transfer 150uL from E1 to F1 |
| F1 | 150uL diluted PicoGreen reagent | Mix and transfer 150uL from F1 to G1 |
| G1 | 150uL diluted PicoGreen reagent | Mix and discard 150uL from G1 |
| H1 | 150uL diluted PicoGreen reagent | Do not add anything |
9. Set microplate reader for excitation 480nm/emission 520nm (or other appropriate pair)
10. Calculate linear relationship of the DNA concentration vs fluorescence of the standard and use to determine amplicon concentration (May delete one or two points from the standard array to get your R-value closer to 1)
- Obtain the linear relationship between the standard concentration and the measurements via a x-y scatter plot made in Excel. Infer calculation of your concentration–> use to calculate concentrations in the spreadsheet
- y= mx + b Solve for x
- Remember to divide the whole equation by the amount of amplicon added ( in this protocol = 2uL )
11. Calculate the volume of amplicon to pool by dividing the amount of amplicon needed by the concentration of the amplicon in the sample
- a) To pool 240 ng of amplicon from a sample at 5.5ng ul^-1; 100ng ul^-1 =18.2uL to get 100 ng of amplicon
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- If over 50, it suggests bad PCR (too much DNA)
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- b)The amount of amplicon needed is arbitrary, though using larger volumes will reduce pipetting variability between samples
12. Pool samples
13. MoBio PCR CleanUp
- Can use 1000uL and 200uL at point 1
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- Can repeat the clean-up to increase concentration
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Finding Amount of DNA to Pool for Engencore
- Excel Sheet of DNA Concentrations
1. Target amount of DNA can be anywhere from 240ng to as low as ~65ng
2. Divide the DNA concentration by 240 ng to get the amount (ul) you would need extract from the well to add to the pooling vial
3. Determine if most of your samples can be pooled based on the calculated amount (amount after calculation can’t be larger than the amount of DNA in the well ex. calculated value is 105uL and the amount of actual DNA in the well is 50uL)
4. If most of the samples calculated amount value are out of range, change the target DNA concentration until you have calculated amount values that incorporate most of the samples and fall within a viable range (no less than 0.5uL and no higher than the amount in the well)
- You want to keep the target amount of DNA to be as high as possible but you want these to include as many samples as possible too
- Keep in mind the calculated amount cannot be lower than 0.5uL
5. Once you find your ideal target amount, pool the viable samples and you are ready for PCR clean-up (if necessary)