Aug 04, 2020
Low-input DNA extractions in 96-well plates
Forked from Honeybee DNA extractions in 96-well plates
- Tom Harrop1,
- Reuben McKay Vercoe1,
- Ciarán Cuddy1
- 1University of Otago
Protocol Citation: Tom Harrop, Reuben McKay Vercoe, Ciarán Cuddy 2020. Low-input DNA extractions in 96-well plates. protocols.io https://dx.doi.org/10.17504/protocols.io.birqkd5w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 20, 2020
Last Modified: August 04, 2020
Protocol Integer ID: 39440
Materials
MATERIALS
3.2 mm stainless steel beads, RNase freeCatalog #NEXSSB32-RNA
96 Well 0.8mL Plate (Bulk)Thermo FisherCatalog #AB0859
ZymoBIOMICS Lysis SolutionZymo ResearchCatalog #D4300-1-150
Quick-DNA Magbead Plus KitZymo ResearchCatalog #D4082
RNase A solution in 10 mM Tris-HCl, pH 7.5, 15 mM NaCl at a concentration of 10 mg/mL:
250 mg RNase A
250 µL 1 M Tris-HCl pH 7.5
75 µL 5 M NaCl
Make up to 25 mL
Proteinase K solution in 50 mM Tris, pH 8, 3 mM CaCl2, 50% Glycerol at a concentration of 20 mg/mL:
100 mg Proteinase K
250 µL 1 M Tris-HCl pH 8
6 µL 2.5 M CaCl2
Make up to 5 mL
Prepare lysate for extraction
Prepare lysate for extraction
Prepare lysate for extraction
Prepare 100 µL lysis buffer per sample.
| Lysis solution | 99 µL | |
| RNase solution (10 mg/mL) | 1 µL | |
| Total | 100 µL |
The Zymo lysis solution can be bought separately.
This protocol also works with the Zymo Solid Tissue Buffer II that is supplied with the Quick-DNA Magbead Plus kit. Solid Tissue Buffer II comes as a 2x concentrate and has to be diluted with nuclease-free water.
Dissect e.g. 1 M. hyperodae pupa for a single sample.
Place up to 88 individuals in a 1000 µL, round well deepwell plate
Add 2 3.2 mm stainless steel ball bearings and 100 µL of lysis buffer with RNase to each well.
Homogenise the tissue for 90 seconds at 1200 RPM using a plate-compatible tissue homogenizer, e.g. SPEX SamplePrep 2010 Geno/Grinder.
00:01:30
Seal the plate and mix on the plate shaker. Make sure the paste is resuspended.
Incubate at 37°C for 30 minutes.
00:30:00
37 °C
Add 5 µL Proteinase K solution (20 mg/mL) and mix on the plate shaker.
Incubate at 55°C for 120 minutes, shaking the plate for one minute every 30 minutes.
02:00:00
55 °C
Spin the plate for 10 minutes at 1,000 RPM.
1000 rpm, 15°C, 00:10:00
Transfer 100 µL of lysate to a 0.8 mL deepwell plate.
DNA extraction
DNA extraction
Set up the reagents from the ZYMO Quick-DNA Magbead Plus Kit
Add the following reagents to the 7-position ReservoirRack
| Reagent | Reservoir volume (mL) | 8 | 16 | 24 | 32 | 40 | 48 | 56 | 64 | 72 | 80 | 88 | |
| 1: 300 µL MBB : 5 µl beads | 30 | 2645 | 5085 | 7525 | 9965 | 12405 | 14845 | 17285 | 19725 | 22165 | 24605 | 27045 | |
| 2: DNA pre-wash buffer | 100 | 3340 | 5340 | 7340 | 9340 | 11340 | 13340 | 15340 | 17340 | 19340 | 21340 | 23340 | |
| 3: g-DNA wash buffer | 100 | 5340 | 9340 | 13340 | 17340 | 21340 | 25340 | 29340 | 33340 | 37340 | 41340 | 45340 | |
| 4: Tris-HCl | 10 | 702 | 902 | 1102 | 1302 | 1502 | 1702 | 1902 | 2102 | 2302 | 2502 | 2702 |
Run the epMotion protocol 88x Quick-DNA Magbead Plus Low Volume 40_6.
Run the protocol with level sensing enabled.