TITLE: DNA extraction from 5uL mouse blood samples (KingFisher Flex 96-well)
AUTHORS: Petra Schneider, Charlotte Repton, Sarah Reece

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>DNA extraction from 5uL mouse blood samples, for subsequent qPCR to detect </span><span style = "font-style:italic;">Plasmodium </span><span> parasites within the blood. Using KingFisher Flex (96 well) system with  </span><a href="https://www.protocols.io/reagents/thermofisher/magmaxtrade96dnamultisamplekit-226703" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">MagMAX™-96 DNA Multi-Sample Kit</span></a><span>. Protocol to import into BindIt Software attached.</span></div></div>

[STEPS]
?. [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).
?. [Sample Collection]
Take a 5 µL blood sample (we use glass capillaries) and dispense the blood immediately into the citrate saline. Repeat for all samples.
?. [Sample Collection]
Mix with pipette and replace lid; or replace lid and mix by carefully tapping the tube.
?. [Sample Collection]
Freeze samples at -20C (short term), -70°C (long-term) or immediately extract DNA.
?. [Prepare for DNA extraction]
Defrost your samples.
?. [Prepare for DNA extraction]
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 for DNA extraction]
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.   ABC1  Reagent    Location    Action  2  Lysis buffer    Bench    None  3  Wash 1    Bench  Add 5.4 mL 100% isopropanol4  Wash 2    Bench  Add 40 mL 100% ethanol5Elution Buffer 1  Bench    None  6Elution Buffer 2  Bench    None  7DNA Binding beads  4 °C fridge    None  8Proteinase K solution  -20 °C freezer    None  9  PK buffer    Bench    None  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!
ABC1  Reagent    Location    Action  2  Lysis buffer    Bench    None  3  Wash 1    Bench  Add 5.4 mL 100% isopropanol4  Wash 2    Bench  Add 40 mL 100% ethanol5Elution Buffer 1  Bench    None  6Elution Buffer 2  Bench    None  7DNA Binding beads  4 °C fridge    None  8Proteinase K solution  -20 °C freezer    None  9  PK buffer    Bench    None
?. [Prepare for DNA extraction]
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 for DNA extraction]
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.
?. [Prepare for DNA extraction]
Fill the plates as shown in the table:  AB1  Plate (type)    Contents /well  2Tip plate(KF)+DWTip Comb  N/A  3  Sample (DW)  50 uL ProK mix (prepared in step 6)  5 uL sample4  Wash 1 (DW)  150 uL wash 1 solution5  Wash 2_1 (DW)  150 uL wash 2 solution6  Wash 2_2 (DW)  150 uL wash 2 solution7  Elution (KF)  75 uL elution buffer 1 Note:  Use protK mix to dilute 5uL blood sample before transfer – easier pipetting.
AB1  Plate (type)    Contents /well  2Tip plate(KF)+DWTip Comb  N/A  3  Sample (DW)  50 uL ProK mix (prepared in step 6)  5 uL sample4  Wash 1 (DW)  150 uL wash 1 solution5  Wash 2_1 (DW)  150 uL wash 2 solution6  Wash 2_2 (DW)  150 uL wash 2 solution7  Elution (KF)  75 uL elution buffer 1
?. [DNA extraction]
Connect the Kingfisher instrument to the computer using USB cable (note: this should already be there).
?. [DNA extraction]
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).
?. [DNA extraction]
Select the protocol (see step 22), and click on the ‘start’ (green arrow) button on the BindIt software.
?. [DNA extraction]
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.
?. [DNA extraction]
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.
?. [DNA extraction]
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.
?. [DNA extraction]
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.
?. [DNA extraction]
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.
?. [DNA extraction]
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]
Optimized machine ProtocolFile to import into your BindIt Software: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):  ABCD1  Step name    Description    Plate    Step end (approx. time from start)  2  Pick up tip         Tip plate  start3  Lysis 1    30s bottom mix  Temperature 20°C    Sample    30s   4  Lysis 2    Heating during mixing 62°C, 10 min paused, tip above well/tube surface, postmix 30s slow.    Sample    11 min   5  Lysis 3    Heating during mixing 62°C, 10 min paused, tip above well/tube surface.    Sample    21 min   6  Add lysis buffer       Dispense.  Message: “Add 200 mL lysis buffer.”    Sample    (~ 3 min)  24 min  7  Lysis 4    3 min 30s bottom mix.  Temperature 20°C    Sample    27 min 30s  8  Add beads    Dispense.  Message: “Add 20 mL beads.”    Sample    (~ 5 min)  32 min 30s  9  Mix beads    3 min bottom mix.  Temperature 20°C    Sample    35 min 30s  10  Add isopropanol    Dispense.  Message: “Add 240 mL ispropanol.”    Sample    (~ 3 min)  38 min 30s  11  Mix isopropanol    5 min half mix; collect beads count 5
  time 30s.  Temperature 20°C    Sample    46 min  12  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  13  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  14  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  15  Dry    Dry time 3 min, outside well/tube.    Wash 2_2    53 min 39s  16  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  17  Add elution buffer 2    Dispense.  Message: “Add 75 mL elution buffer 2.”    Elution    (~ 3 min)  66 min 9s  18  Elution 2    30s bottom mix, 2 min medium; collect
  beads count 5, time 30s.  Temperature 20°C    Elution    71 min 9s  19  Leave tip in wash 2_2.         Wash 2_2
ABCD1  Step name    Description    Plate    Step end (approx. time from start)  2  Pick up tip         Tip plate  start3  Lysis 1    30s bottom mix  Temperature 20°C    Sample    30s   4  Lysis 2    Heating during mixing 62°C, 10 min paused, tip above well/tube surface, postmix 30s slow.    Sample    11 min   5  Lysis 3    Heating during mixing 62°C, 10 min paused, tip above well/tube surface.    Sample    21 min   6  Add lysis buffer       Dispense.  Message: “Add 200 mL lysis buffer.”    Sample    (~ 3 min)  24 min  7  Lysis 4    3 min 30s bottom mix.  Temperature 20°C    Sample    27 min 30s  8  Add beads    Dispense.  Message: “Add 20 mL beads.”    Sample    (~ 5 min)  32 min 30s  9  Mix beads    3 min bottom mix.  Temperature 20°C    Sample    35 min 30s  10  Add isopropanol    Dispense.  Message: “Add 240 mL ispropanol.”    Sample    (~ 3 min)  38 min 30s  11  Mix isopropanol    5 min half mix; collect beads count 5
  time 30s.  Temperature 20°C    Sample    46 min  12  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  13  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  14  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  15  Dry    Dry time 3 min, outside well/tube.    Wash 2_2    53 min 39s  16  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  17  Add elution buffer 2    Dispense.  Message: “Add 75 mL elution buffer 2.”    Elution    (~ 3 min)  66 min 9s  18  Elution 2    30s bottom mix, 2 min medium; collect
  beads count 5, time 30s.  Temperature 20°C    Elution    71 min 9s  19  Leave tip in wash 2_2.         Wash 2_2
?. [Sample Collection]
Spin down and remove supernatant. Centrifuge 3 minutes at 4ºC. Remove supernatant, using clean pipette tip for each sample.