A | B | C | D | E | F | |
PotatoMASH step and supplies | Provider / code | pack price | pack units | preps | sample costs (€) | |
Template DNA normalization: | ||||||
Quant-iT™ PicoGreen® dsDNA Assay Kit | Thermofisher P7589 | 510 | 2000 | 1916 | 0.266 | |
Nunc™ F96 MicroWell™ Black Plates | Thermofisher 236105 | 112 | 50 | 4400 | 0.025 | |
10µL tips for quantitation | Greiner 771290 | 9 | 1000 | 1000 | 0.009 | |
200µL tips for quantitation | Sarstedt 70.760.002 | 40 | 10000 | 10000 | 0.004 | |
10µL filter tips for normalization | Sarstedt 70.1130.210 | 70 | 1920 | 1920 | 0.036 | |
96 well PCR plate | Thermo Sci. 10425733 | 58.27 | 25 | 2400 | 0.024 | |
plate adhesive lid | Greiner 676001 | 14.3 | 100 | 9600 | 0.001 | |
PotatoMASH PCR1: | ||||||
QIAGEN Multiplex PCR Plus Kit (100) | Qiagen 6152 | 185 | 2.55mL | 700 | 0.264 | |
PotatoMASH Primers (n=348, 750uM each) | IDT | 5133 | 20µL | 85714 | 0.06 | |
96 well PCR plate | Sarstedt 72.1978.202 | 69.75 | 25 | 2400 | 0.029 | |
10µL filter tips | Sarstedt 70.1130.210 | 70 | 1920 | 1920 | 0.036 | |
Adhesive aluminium foil plate lid | Sarstedt 95.1995 | 50.5 | 100 | 9600 | 0.005 | |
PotatoMASH PCR2: | ||||||
100µL filter tips to dilute PCR1 | Sarstedt 70.760.212 | 70.8 | 1920 | 1920 | 0.037 | |
QIAGEN Multiplex PCR Plus Kit (100) | Qiagen 6152 | 185 | 2.55mL | 490 | 0.378 | |
i5 and i7 Primers (n=96+8) at 100µM | IDT | 667 | 300µL | 2875 | 0.232 | |
96 well PCR plate | Sarstedt 72.1978.202 | 69.75 | 25 | 2400 | 0.029 | |
10µL filter tips | Sarstedt 70.1130.210 | 70 | 1920 | 960 | 0.073 | |
Adhesive aluminium foil plate lid | Sarstedt 95.1995 | 50.5 | 100 | 9600 | 0.005 | |
Library normalization, Pooling wells and Concentration-purification: | ||||||
SequalPrep™Normalization Plate Kit | Invitrogen A1051001 | 1050 | 10 | 960 | 1.094 | |
10µL tips for binding step | Greiner 771290 | 9 | 1000 | 500 | 0.018 | |
200µL tips for washing step | Sarstedt 70.760.002 | 40 | 10000 | 10000 | 0.004 | |
200µL tips for elution step | Sarstedt 70.760.002 | 40 | 10000 | 10000 | 0.004 | |
plate adhesive lid | Greiner 676001 | 14.3 | 100 | 9600 | 0.001 | |
Size selection, Purification, Quantification and Pooling plates: | ||||||
Buffer PB | Qiagen 19066 | 87 | 500mL | 6400 | 0.014 | |
15mL tubes | Sarstedt 62.554.002 | 55 | 500 | 48000 | 0.001 | |
QIAquick PCR purification Kit | Qiagen 28704 | 104.14 | 50 | 4800 | 0.022 | |
1mL tips | Fisherbrand 11548442 | 8.92 | 1000 | 6857 | 0.001 | |
Wizard SV Gel and PCR Clean-Up System | Promega A9281 | 94 | 50 | 4800 | 0.02 | |
AMPure XP magnetic beads | Beckman C. A63881 | 1326 | 60mL | 57600 | 0.023 | |
100µL filter tips | Sarstedt 70.760.212 | 70.8 | 1920 | 30720 | 0.002 | |
Qubit™ dsDNA BR Assay Kit | Thermofisher Q32853 | 275 | 500 | 48000 | 0.006 | |
Qubit™ assay tubes | Thermofisher Q32856 | 70 | 500 | 48000 | 0.001 |
A | B | C | D | |
Concentration of standard (λ) | Volume of 1X TE | Volume and concentration of Lambda | Final DNA Concentration PicoGreen Assay | |
2000 ng/ml | 1470 μL | 30 μL of 100 μg/mL λ | 1000 ng/ml | |
200 ng/ml | 1350 μL | 150 μL of 2000 ng/mL λ | 100 ng/ml | |
20 ng/ml | 1350 μL | 150 μL of 200 ng/mL λ | 10 ng/ml | |
2 ng/ml | 1350 μL | 150 μL of 20 ng/mL λ | 1 ng/ml |
A | B | C | |
Reagent | 1 x 96-well plate | 4 x 96-well plates | |
20X TE | 500 µl | 2 ml | |
Autoclaved dd-water | 9.5 ml | 38 ml | |
Total | 10 ml | 40 ml |
A | B | |
Column 12 | Content | |
12A | 2000 µg/ml λ | |
12B | 200 µg/ml λ | |
12C | 20 µg/ml λ | |
12D | 2 µg/ml λ | |
12E | 1X TE | |
12F | Empty | |
12G | Empty | |
12H | Empty |
A | B | C | |
Reagent | 96-well plate | 4 x 96-well plates | |
1X TE (from step 1.2) | 9.95 ml | 39.8 ml | |
PicoGreen reagent | 50 µl | 200 µl |
A | B | C | |
Reagent | Final content per prep | Cocktail for 96-well plate | |
Nuclease free water | 0.1 µl | 10 µl | |
PotatoMASH primers 125nM each primer (A pool of 678 primers=339 primer pairs) | 1.4 µl (25nM each primer) | 140 µl | |
Qiagen Plus multiplex master mix (2x, ref:206152) | 3.5 µl (1x) | 350 µl | |
DNA (20ng/µl) | 2 µl (40ng) | - | |
Total | 7 µl | 500 µl (5 µl per sample) |
A | B | C | D | E | F | G | H | |
Step | Cycles | Temp | Time | Annealing Temp | Time | Elongation Temp | Time | |
Initial Denaturation | 1x | 95 °C | 15 min | |||||
Initial amplification | 8x | 95 °C | 30 sec | 0.2°C/sec ramp down to 57°C | 30 sec | 72 °C | 2 min | |
Amplicon multiplication | 16x | 95 °C | 30 sec | 65 °C | 30 sec | 72 °C | 30 sec | |
Hold | 1x | 10 °C | ∞ |
A | B | C | D | E | F | G | H | |
Step | Cycles | Temp | Time | Annealing Temp | Time | Elongation Temp | Time | |
Initial Denaturation | 1x | 95 °C | 15 min | |||||
Initial amplification | 5x | 95 °C | 30 sec | 0.2°C/sec ramp down to 57°C | 30 sec | 72 °C | 2 min | |
Amplicon multiplication | 10x | 95 °C | 30 sec | 65 °C | 30 sec | 72 °C | 30 sec | |
Hold | 1x | 10 °C | ∞ |
A | B | C | |
Reagent | Final content per prep | Cocktail for 96-well plate | |
Plate-specific i7 10 µM primers | 1 µl | 100 µl | |
206152 | 5 µl | 500 µl | |
Well-specific i5 10 µM primers | 1 µl | - | |
PCR1 product diluted 1:15 | 3 µl | - | |
Total | 10 µl | 600 µl (6 µl per sample) |
A | B | C | D | E | F | G | H | |
Step | Cycles | Temp | Time | Annealing Temp | Time | Elongation Temp | Time | |
Initial Denaturation | 1x | 95 °C | 15 min | |||||
Amplification cycles | 10x | 98 °C | 10 sec | 65 °C | 30 sec | 72 °C | 30 sec | |
Final Extension | 1x | 72 °C | 5 min | |||||
Hold | 1x | 10 °C | ∞ |
A | B | C | |
Reagent | 1 sample | 7 samples | |
Nuclease-free water | 3 µl | 21 µl | |
Qiagen Master Mix (2x) | 5 µl | 35 µl | |
Illumina primers 10 µM (mix of both, 5µM each) | 1 µl | 7 µl | |
library (0.1 ng/ul) | 1 µl | - | |
Total | 10 µl | 63 µl (9 µl per sample) |
A | B | C | D | E | F | |
Step | Cycles | Denaturation Temp | Time | Annealing and Elongation Temp | Time | |
Initial Denaturation | 1x | 90 °C | 1 min | |||
Annealing & Elongation | 16x | 95 °C | 15 sec | 63 °C | 45 sec | |
Hold | 1x | 10 °C | ∞ |