Thermo Fisher Scientific PrepSEQ Quick Reference
QUICK REFERENCE
PrepSEQ™ Residual DNA Sample Preparation Kit
For (Genomic DNA) CHO, E. coli, HEK293, Human, MDCK, NSO, Pichia, Sf9 and Baculovirus, and Vero
For (Plasmid DNA) Kanamycin resistance
Catalog Numbers 4402085, 4458435, A46014, A26366, 4464335, 4458441, 4464336, A46066, A41797, A50337
Pub. No. 4469839 Rev. G
Note: For safety and biohazard guidelines, see the “Safety” appendix in the PrepSEQ™ Sample Preparation Kits User Guide
(Pub. No. 4469838). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves.
Prepare the reagents: before first use of the kit .................................................................. 1
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Prepare reagents: before each use of the kit ..................................................................... 1
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Manual protocol for DNA/RNA extraction ....................................................................... 2
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Automated protocol for DNA/RNA extraction .................................................................... 4
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Limited product warranty .................................................................................... 5
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Prepare the reagents: before first use of the kit
Magnetic beads
1. Set a block heater to 37°C.
2. Incubate the Magnetic Particle suspension at 37°C for a minimum of 10 minutes with intermittent vortexing at setting #7, or until the
particles are completely suspended.
Binding Solution
1. Add 30 mL of 100% isopropanol to the Binding Solution bottle.
2. Label the bottle to indicate that it contains isopropanol, then store the bottle at ambient temperature.
Wash Buer Concentrate
1. Add 74 mL of 95% ethanol to one bottle of PrepSEQ™ Wash Buer Concentrate, then mix completely.
2. Label the bottle to indicate that it contains ethanol, then store the bottle at room temperature.
Prepare reagents: before each use of the kit
Proteinase K (PK) mix
• Use Proteinase K (PK) Buer II for new manual protocols and automated protocols.
Note: Proteinase K (PK) Buer is also provided in the kit for use with existing manual protocols that have been internally validated
with this buer.
• Prepare a fresh mix before each use of the kit.
• Include a 10% overage to account for pipetting losses.
For Research Use Only. Not for use in diagnostic procedures.
Component
1 7 10 13 25
Proteinase K, 20 mg/mL 10 μL 70 μL 100 μL 130 μL 250 μL
Number of extractions
Proteinase K (PK) Buer II or Proteinase K
(PK) Buer
60 μL 420 μL 600 μL 780 μL 1,500 μL
Lysis solution
• Prepare a fresh mixture immediately before use or during Proteinase K incubation.
• Prepare 360 µL (amount required) of lysis solution mix per sample.
Reagent Volume for ~20 extractions
Glycogen, 5 mg/mL 180 µL
Yeast tRNA, 10 mg/mL 4 µL
Lysis Buer 7,600 µL
Total 7,784 µL
Manual protocol for DNA/RNA extraction
(Plasmid samples only) Add Yeast tRNA
Plasmid samples require the use of Yeast tRNA as a carrier during the extraction process.
1. Dilute the Yeast tRNA.
Table 1 Diluted Yeast tRNA
Component
Yeast tRNA (10mg/mL) 5 μL
PBS (1X), pH 7.2 245 μL
Total 250 μL
Volume
2. Add 5 μL Diluted Yeast tRNA to 370 μL of each test sample before extraction. This is sucient for triplicate 100 μL extractions.
Digest the test samples and controls
1. Set a block heater to 56°C. If available, set a second block heater to 70°C.
2. Label 2‑mL Safe-Lock tubes:
• 3 for each sample
• 3 for each sample + ERC
• 3 for NEG
3. Add 100 µL of sample, sample + ERC, or 1X PBS to into each tube.
Note: Ensure that Diluted Yeast tRNA was added to each plasmid sample.
4. Add 10 µL of 5 M NaCl and 70 μLProteinase K/Proteinase K Buer II mix.
5. Briefly vortex and centrifuge.
6. Incubate at 56°C for 30 minutes.
If only one block heater is available, after this incubation step is complete, reset the block heater to 70°C for the elution step.
Note: For samples with high protein concentration, extending the incubation time to 60 minutes can increase recovery.
2 PrepSEQ
™
Residual DNA Sample Preparation Kit Quick Reference
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