Nov 11, 2019
DNA extraction from 5uL mouse blood samples (KingFisher Flex 96-well)
- Petra Schneider1,
- Charlotte Repton1,
- Sarah Reece1
- 1University of Edinburgh
Protocol Citation: Petra Schneider, Charlotte Repton, Sarah Reece 2019. DNA extraction from 5uL mouse blood samples (KingFisher Flex 96-well). protocols.io https://dx.doi.org/10.17504/protocols.io.86fhzbn
Manuscript citation:
Schneider, P. et al. (2018) ‘Adaptive plasticity in the gametocyte conversion rate of malaria parasites’, PLoS Pathogens, 14(11). doi: 10.1371/journal.ppat.1007371.
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: November 08, 2019
Last Modified: November 11, 2019
Protocol Integer ID: 29607
Keywords: DNA extraction, mouse, blood, Plasmdium
Abstract
DNA extraction from 5uL mouse blood samples, for subsequent qPCR to detect Plasmodium parasites within the blood. Using KingFisher Flex (96 well) system with MagMAX™-96 DNA Multi-Sample Kit. Protocol to import into BindIt Software attached.
Guidelines
Please note that DNA was extracted using the semi-automatic Kingfisher Flex Magnetic Particle Processor and the MagMax 96-DNA multisample kit for DNA (4413021/4413022Thermo Fisher Scientific) but with adjustments to the manufacturer's protocols (standard protocols 4413021DWblood) to improve recovery.
Materials
MATERIALS
MagMAX™-96 DNA Multi-Sample KitThermo FisherCatalog #4413021
KingFisher™ Flex™ Systems Consumables, KingFisher Flex Microtiter Deepwell 96 plate, sterileThermo FisherCatalog #95040460
KingFisher™ Flex™ Systems Consumables, KingFisher 96 tip comb for DW magnetsThermo FisherCatalog #97002534
KingFisher™ Flex™ Systems Consumables, KingFisher 96 KF microplate (200µL)Thermo FisherCatalog #97002540
In addition you will need: isopropanol, absolute ethanol, citrate saline (0.85 % w/v NaCl, 1.5% w/v trisodium citrate dihydrate) and general lab materials (plastics, pipettes, pipette tips)
Sample Collection
Sample Collection
Dispense 150 µL of citrate saline (0.85 % w/v NaCl, 1.5% w/v trisodium citrate dihydrate) into 0.2 mL strip tubes or 96-well plates, one well per sample. Cover tubes with lids, or plate with a plastic film (the thick version, not the optical film for qPCRs – that one will NOT come off).
Take a 5 µL blood sample (we use glass capillaries) and dispense the blood immediately into the citrate saline. Repeat for all samples.
Mix with pipette and replace lid; or replace lid and mix by carefully tapping the tube.
Spin down and remove supernatant. Centrifuge 3 minutes at 4ºC. Remove supernatant, using clean pipette tip for each sample.
Freeze samples at -20C (short term), -70°C (long-term) or immediately extract DNA.
Prepare for DNA extraction
Prepare for DNA extraction
Defrost your samples.
Get required plastics. You will need 4 Kingfisher DeepWell (DW; Cat# 95040460) plates, 2 Kingfisher 96 (KF; Cat# 97002540) plates and 1 DW tip comb (Cat# 97002534).
Prepare reagents in kit only if opening a new MagMax 96-DNA multisample kit (Cat# 4413021), Add the appropriate amount of ethanol or isopropanol to the wash buffers (see table below) and mark the bottles clearly by crossing out the word “concentrate”, ticking the small checkbox and writing your name/initials and the date.
| Reagent | Location | Action | |
| Lysis buffer | Bench | None | |
| Wash 1 | Bench | Add 5.4 mL 100% isopropanol | |
| Wash 2 | Bench | Add 40 mL 100% ethanol | |
| Elution Buffer 1 | Bench | None | |
| Elution Buffer 2 | Bench | None | |
| DNA Binding beads | 4 °C fridge | None | |
| Proteinase K solution | -20 °C freezer | None | |
| PK buffer | Bench | None |
New MagMax 96-DNA multisample kit - Preparation of reagents (Thermofisher 4413021)
Note: some of the bench-top reagents crystallise when the ambient temperature is low (below ~18°C). To combat this, set a heat block to 25°C before beginning and place the room temperature reagents on it until ready to use, remembering to mix well!
Prepare Proteinase K (ProK) mix. In a sterile, DNase/RNase-free eppendorf tube or vial mix 8 mL of ProK solution and 42uL of PK buffer (total = 50 mL) per well (i.e. sample), plus 10% extra for errors.
Prepare bead mix. In a sterile, DNase/RNase-free eppendorf tube, mix 16 mL of DNA binding beads and 4 mL of nuclease free water (total = 20 mL) per well (i.e. sample), plus 10% extra for errors.
Fill the plates as shown in the table:
| Plate (type) | Contents /well | |
| Tip plate(KF)+DWTip Comb | N/A | |
| Sample (DW) | 50 uL ProK mix (prepared in step 6) 5 uL sample | |
| Wash 1 (DW) | 150 uL wash 1 solution | |
| Wash 2_1 (DW) | 150 uL wash 2 solution | |
| Wash 2_2 (DW) | 150 uL wash 2 solution | |
| Elution (KF) | 75 uL elution buffer 1 |
Plate filling instructions
Note: Use protK mix to dilute 5uL blood sample before transfer – easier pipetting.
DNA extraction
DNA extraction
Connect the Kingfisher instrument to the computer using USB cable (note: this should already be there).
Open the BindIt software. Select ‘Your Kingfisher Machine’ from the drop-down ‘connect’ list to connect to your Kingfisher instrument (note: connection has likely established automatically).
Select the protocol (see step 22), and click on the ‘start’ (green arrow) button on the BindIt software.
Load the plates into the machine. The machine will turn the turntable and prompt you to load the plates in the correct order. Press the ‘start’ button on the instrument to confirm you have loaded the current plate, this tells the machine to progress to the next plate. Make sure to line up the A1 well of the plate with the marked corner on the turntable position.
Follow dispense step instructions. The run will begin once all plates are loaded. The instrument will prompt you to add the correct reagents at the dispense steps (grey cells in machine protocol, below). The first dispense step is roughly 20min after start-time. A guide to the approximate length of time until a step ends (so that you can work out how long between dispense steps) is also included in the machine protocol table below.
Remove plates when the run has ended (as prompted by machine). Export AND SAVE the run details as a PDF (automatically opened in the software) – it contains details of the times each steps were begun as well as temperatures at frequent intervals, if something has gone wrong this may give you the answer why. Do not yet discard.
Check, seal and label DNA extracts. Check there is liquid in the elution plate (if not, something has gone wrong!). Seal the elution plate with a plastic film (make sure it is freezer-suitable and do not use the optical qPCR films – these do NOT come off) and label it carefully.
Freeze at -20°C or use immediately for qPCR. For use in qPCRs, we recommend diluting at least 1 volume extracted DNA + 2 volumes water to remove the effects of inhibitors.
Discard all plates, except the elution plate. If you haven’t used every well, you can keep them to be reused (be sure to use different, clean wells to the ones you have already used). And, if you do keep them for a while – remove liquids from used wells before storage.
Kingfisher Protocol
Kingfisher Protocol
Optimized machine Protocol
File to import into your BindIt Software:
DNA5uLMouseBlood.bdz
DNA5uLMouseBlood.bdz Summary:
Plates (add to BindIt software under the ‘Layout’ tab):
- Tip plate (KF 96 plate with DW 96 tip comb inserted)
- Sample plate (DW microtiter plate)
- Wash 1 (DW microtiter plate)
- Wash 2_1 (DW microtiter plate)
- Wash 2_2 (DW microtiter plate)
- Elution (KF 96 plate)
Protocol (add to BindIt software under the ‘Protocol’ tab):
| Step name | Description | Plate | Step end (approx. time from start) | |
| Pick up tip | Tip plate | start | ||
| Lysis 1 | 30s bottom mix Temperature 20°C | Sample | 30s | |
| Lysis 2 | Heating during mixing 62°C, 10 min paused, tip above well/tube surface, postmix 30s slow. | Sample | 11 min | |
| Lysis 3 | Heating during mixing 62°C, 10 min paused, tip above well/tube surface. | Sample | 21 min | |
| Add lysis buffer | Dispense. Message: “Add 200 mL lysis buffer.” | Sample | (~ 3 min) 24 min | |
| Lysis 4 | 3 min 30s bottom mix. Temperature 20°C | Sample | 27 min 30s | |
| Add beads | Dispense. Message: “Add 20 mL beads.” | Sample | (~ 5 min) 32 min 30s | |
| Mix beads | 3 min bottom mix. Temperature 20°C | Sample | 35 min 30s | |
| Add isopropanol | Dispense. Message: “Add 240 mL ispropanol.” | Sample | (~ 3 min) 38 min 30s | |
| Mix isopropanol | 5 min half mix; collect beads count 5 time 30s. Temperature 20°C | Sample | 46 min | |
| Wash 1 | Release beads 30s bottom mix; [mix 10s half mix, mix 10s fast] x 3 loops; collect beads count 3, time 1s. Temperature 20°C | Wash 1 | 47 min 33s | |
| Wash 2_1 | Release beads 30s bottom mix; [mix 10s half mix, 10s fast] x 3 loops; collect beads count 3, time 1s. Temperature 20°C | Wash 2_1 | 49 min 6s | |
| Wash 2_2 | Release beads 30s bottom mix; [mix 10s half mix, 10s fast] x 3 loops; collect beads count 3, time 1s. Temperature 20°C | Wash 2_2 | 50 min 39s | |
| Dry | Dry time 3 min, outside well/tube. | Wash 2_2 | 53 min 39s | |
| Elution 1 | Release beads 30s bottom mix; heating during mixing 72°C, preheat, mix 5 min slow; postmix 4 min fast. | Elution | 63 min 9s | |
| Add elution buffer 2 | Dispense. Message: “Add 75 mL elution buffer 2.” | Elution | (~ 3 min) 66 min 9s | |
| Elution 2 | 30s bottom mix, 2 min medium; collect beads count 5, time 30s. Temperature 20°C | Elution | 71 min 9s | |
| Leave tip in wash 2_2. | Wash 2_2 |
KingFisher Protocol for DNA extraction from 5uL mouse blood samples (BindIt software) with added time frame for extraction run.