TITLE: Insects and DNA metabarcoding - InsectMobile laboratory protocols 
AUTHORS: Cecilie Svenningsen, Lene Bruhn Pedersen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This document is a collection of laboratory protocols for the citizen science and DNA metabarcoding research project 'InsectMobile' at the Natural History Museum of Denmark/University of Copenhagen 2017-2021. The protocol(s) cover all methodological steps from sample pre-processing to library build for next-generation-sequencing on a HiSeq 4000 150 bp PE platform.</div><div class = "text-block">Samples are collected with car rooftop-mounted insect nets by citizen scientists in the month of June 2018-2020 and the detachable end of the net containing flying insects is shipped to the laboratories at the Natural History Museum of Denmark/GLOBE Institute in containers containing 96% ethanol. </div><div class = "text-block">The insects are then size sorted, the large size fraction is identified by morphology and counted, and the sample dry-weight is obtained after which the bulk samples are processed with a DNA metabarcoding protocol. Morphological identification of the insects is not covered by this protocol.   </div></div>

[STEPS]
?. [Purifying digest]
Purify the  aliquots in the QIASymphony robot. 24 samples can be purified each time and always include one extraction blank aliquot as a robot blank. The robot runs 24 samples in one hour. The protocol draws on the following resources:·DNA Handbook: EN-QIAsymphony-DNA-Handbook.pdf·Low content purification protocol: HB-0977-S09_002_PS_QS_LowHighContent_V7_DSP_DNA_UV_0818_WW.pdf·General description of the QIASymphony SP/AS robot: QIASymphony_SPAS_GeneralDescription.pdf·User manual: QIAsymphony_SPAS_User_Manual_Operating_the_QIAsymphony_SP_1211.pdf General information200 µl tip boxes for QIACube works in QIASymphony (QS), but not the 1000 µl tips. QIASymphony uses 1500 µl tips (Cat No./ID: 997024)On QIAGEN’s webpage search for instrument for instrument information and kit for kit informationOn the specific kit webpage, one can find handbooks for the kit under the tab ‘ressources’The document called ‘labware test’ shows, which protocol and labware are recommended for a specific application/kit. Green = good, Yellow = has not been tested, red = not recommendedQIAGEN can help with a custom protocol if a custom elution is required Every time you open the hood the machine asks you whether you have changed anythingTip chute can be cleaned with normal dishwashing detergent if neededDrop catchers should be checked occasionally for spillage and cleaned with dishwashing detergent and 70% EtOH if neededTips tend to stack in the waste bag and this can cause malfunction, so kick the bag occasionally to make sure they are evenly distributedDO NOT USE EtOH ON PLEXIGLASS & THE SCREEN!Change the O-rings every month to keep the pipetting preciseYou can work in the next drawer while the machine checks the previous drawerAll catalog numbers are according to QIAGENS webpage (26.11.2018).
230 µl
?. [Purifying digest]
When loading the robot you always go from the end (elution) to the beginning (samples) (right to left).
?. [Purifying digest]
Choose the tab ‘Sample preparation’ for running samples
?. [Elution drawer]
An adaptor = a cooling rack and you use EMTR plates for eluation (96 tubes/samples, deep-well (special tube racks required)) OR single tubes (24 samples). Elution slot 1 is the only slot with cooling.
?. [Elution drawer]
If you use single tubes, label them and place them in the rack without lids on
?. [Elution drawer]
When placing the cooling rack, make sure the marked ‘A’ on the plate is placed at the ‘A’ mark on the drawer – THIS IS IMPORTANT for knowing which samples are which and the old racks can be placed both ways. For 96 plates: When the bottom is removed from the rack the lid should be on
?. [Elution drawer]
Adaptor holes in the bottom show how it should be placed on slot 1 (=cold, )
4 °C
?. [Elution drawer]
Press ‘ID’ and register the type of rack you are using - use the document ‘labware test’ because the plate should match the adaptor and to run properly the robot needs to know which type it is
?. [Elution drawer]
Press ‘Rack ID’ to give the rack a name
?. [Elution drawer]
Press ‘OK’. Now the system will check whether the drawer has been loaded correctly
?. [Waste drawer]
Make sure there are waste buckets in the empty slots and remove the lid from partially filled waste buckets
?. [Waste drawer]
Make sure there is room in the bag for used tips. Waste drawer layout:
?. [Waste drawer]
Press ‘OK’. The robot will check the ‘Waste’ drawer and the bucket closest to you, which needs to be empty if the machine is turned off. If the bucket is full then place a lid on it and throw it out
?. [Reagents and consumables drawer]
You can run two kits, even different kits, at the same time. The robot will let you know prior to a run, whether you need to load more consumables, based on the number of samples. When restocking consumables, use the whole unit box to minimize the risk of contamination. You can scan specific slots for the drawer, which comes in handy if you only make changes to e.g. tip stocks.Load tips, cartridges and rod covers, if needed.
?. [Reagents and consumables drawer]
Make sure the orientation of the tip holders is correct. You do not need to put tips in a specific slot for the machine to work.
?. [Reagents and consumables drawer]
Vortex the beads trough for min. 3 minutes to re-suspend the beads. The lid can be greasy when you take it off, and make sure to wipe the barcode of if it is dirty, so the machine can read it
?. [Reagents and consumables drawer]
Follow the protocol in the kit. If the reagents are unused, a piercing lid has to be placed on the reagent cartridge. Be careful – the piercing lid is sharp
?. [Reagents and consumables drawer]
The original lid can be re-used to hold the sealing lids if you are re-using a reagent cartridge
?. [Reagents and consumables drawer]
Mount the enzyme rack on the side of the reagent cartridge. Lids can be placed in the holders below the enzyme rack
?. [Reagents and consumables drawer]
Load the reagent cartridge
?. [Reagents and consumables drawer]
Let the robot check whether you loaded the drawer correctly. This check takes a long time (5-10 min)
?. [Sample drawer]
The robot uses four-channel pipettes, so make sure everything is loaded in four, e.g. if you have vials of different size, then they need to be loaded in fours. The 3B adapter in the sample racks is used for the Sarstedt microtubes.
?. [Sample drawer]
Remove the lids from the sample tube and place them in the sample rack. NOTE THE ORDER OF THE SAMPLES
?. [Sample drawer]
Load the sample rack in a lane by supporting the rack below with one hand and slide the rack to the first line and wait until the camera comes out
?. [Sample drawer]
When the light blinks, push the rack steadily into the robot and make sure the robot has registered all the tubes (this step take practice and you might need to reload it several times). The robot tells you if you need to reload the samples
?. [Sample drawer]
Choose the batch on the screen to assign names to your samples and specify the protocol and vial type
?. [Sample drawer]
Select all samples and assign the tube type. We use the vial holder 3B and the tubes are called Sarstedt screw skirted 72.694(.006)
?. [Sample drawer]
Unless you use a barcode reader, you will need to manually assign names to each sample in the system. Choose each sample and press ‘sample id’ to assign a name
?. [Sample drawer]
Choose your setup and which protocol you use (InsectMobile uses ‘DNA Tissue LC’). Press “Next”
?. [Sample drawer]
Press “Queue” and then “Run”
?. [Sample drawer]
The robot might tell you that you need to load more tips, rod covers or sample prep cartridges. You can specify which consumables that that should be scanned after you make the changes
?. [Sample drawer]
If you use the USB stick, then transfer the run file by pressing “Tools”→”User Management”→”File transfer”→”Save to USB”→”Results file”→”Transfer”
?. [After the last run of the day & general cleaning]
See ‘End of working day’ sheet in the yellow plastic pocket next to the machine (you do not need to follow general description maintenance instructions unless there has been a spill).
?. [After the last run of the day & general cleaning]
Clean QS every Friday (if it has been in use) by removing all plastic in the drawers and turning on the UV-light for half an hour.
?. [After the last run of the day & general cleaning]
Change the O-rings every month (kit and protocol can be found under the machine)
?. [Diluting DNA for standard input in PCR]
Quantify the DNA concentration in each sample with QuBit HS (BR if too high).  Use the template to calculate how much DNA and molecular grade water should be used for a final volume of   with an approximate DNA concentration of  Make the dilutions in 8-strip PCR tubes and include a blank (molecular grade water) in every 6th row. PCRs can then be carried out for full libraries of 96 samples in each library.Materials neededMolecular grade water aliquotPCR strips20 ul or 200 ul empty tip container for holding stripsPermanent marker for writing on PCR tubes (IM_numbers)10 or 20 ul tips200 ul tips200 and 20/10 ul pipettesRack for water aliquot
50 µl
2
?. [Diluting DNA for standard input in PCR]
In the “Dilutions” sheet input the PCRID and associated QuBit measurement. Input HS if this worked otherwise input BR.
?. [Diluting DNA for standard input in PCR]
If your sample is too low for QuBit to perform a measurement then put the final volume in the column DNA added
?. [Diluting DNA for standard input in PCR]
Generally, we want a final volume of . However, this is not always possible. If you sample measurement is below  but it is not too low, then still treat it as a too low sample and add the finale volume in the column DNA added
50 µl
2
?. [Diluting DNA for standard input in PCR]
If your sample has a very high DNA concentration then you need to increase the finale volume → try to get the volume around 1 in the column DNA added
?. [tagged PCR]
When the samples have been extracted, purified, measured with Qubit and diluted to 2 ng/µL, they are ready to be amplified by polymerase chain reaction (PCR). Ideally, PCR setup prior to DNA input should be carried out in a DNA-free lab. We set up the MM and add the tagged primers in the DNA-free prior to adding the 2 ng/µL sample in DNA lab.Setup your PCR spreadsheet prior to entering the lab and print it. It should contain the tag combination of your primers for each tube, which samples go in each tube and the calculated reaction volume of Master Mix (MM). See attached example of PCR spreadsheets setup and thermocycler settings. Example setup for the fwh primer pair (COI):  , example setup for the art primer pair (COI):  , example setup for the ins primer pair (16S): Remember to add a couple of samples to the sample size, so you make enough MM, e.g. if you have 24 samples add 2 more so you make MM for 26 samples. The tag combination of each sample allows us to identify the origin of the sample when we get our sequencing results, so it is important it is consistent or else we will not be able to identify the sample in the data analysis. Switch tag combination for each library by either jumping 8 tags on the reverse or the forward primer (be consistent), since it makes it easier using the multichannel pipette.Materials needed70% EtOH 1:20 Bleach Waste binPipette holderPipettes of the volume needed (see recipe/spreadsheet)Multichannel pipetteRepeater (electronic pipette)Pipette tip boxes that match the pipettesPipette tip for the repeaterPrimers from the freezerdNTPs in the correct dilution BSA MgCl2PCR bufferTag Gold polymerase Molecular grade H2O aliquot in 50 mL tubePCR tubes in strips of 8 with individual lids2 x Eppendorf tubes for MM & H2OFreezing rack for PCR buffer reagents, Master Mix (MM) and H2OFreezing rack for PCR tube stripsTwo rack for primersPermanent marker1:20 Bleach Waste binPipette holderPipettes of the volume needed (see recipe/spreadsheet)Multichannel pipetteRepeater (electronic pipette)Pipette tip boxes that match the pipettesPipette tip for the repeaterPrimers from the freezerdNTPs in the correct dilution BSA MgCl2PCR bufferTag Gold polymerase Molecular grade H2O aliquot in 50 mL tubePCR tubes in strips of 8 with individual lids2 x Eppendorf tubes for MM & H2OFreezing rack for PCR buffer reagents, Master Mix (MM) and H2OFreezing rack for PCR tube stripsTwo rack for primersPermanent markerWaste binPipette holderPipettes of the volume needed (see recipe/spreadsheet)Multichannel pipetteRepeater (electronic pipette)Pipette tip boxes that match the pipettesPipette tip for the repeaterPrimers from the freezerdNTPs in the correct dilution BSA MgCl2PCR bufferTag Gold polymerase Molecular grade H2O aliquot in 50 mL tubePCR tubes in strips of 8 with individual lids2 x Eppendorf tubes for MM & H2OFreezing rack for PCR buffer reagents, Master Mix (MM) and H2OFreezing rack for PCR tube stripsTwo rack for primersPermanent markerBSA MgCl2PCR bufferTag Gold polymerase Molecular grade H2O aliquot in 50 mL tubePCR tubes in strips of 8 with individual lids2 x Eppendorf tubes for MM & H2OFreezing rack for PCR buffer reagents, Master Mix (MM) and H2OFreezing rack for PCR tube stripsTwo rack for primersPermanent markerMgCl2PCR bufferTag Gold polymerase Molecular grade H2O aliquot in 50 mL tubePCR tubes in strips of 8 with individual lids2 x Eppendorf tubes for MM & H2OFreezing rack for PCR buffer reagents, Master Mix (MM) and H2OFreezing rack for PCR tube stripsTwo rack for primersPermanent markerMolecular grade H2O aliquot in 50 mL tubePCR tubes in strips of 8 with individual lids2 x Eppendorf tubes for MM & H2OFreezing rack for PCR buffer reagents, Master Mix (MM) and H2OFreezing rack for PCR tube stripsTwo rack for primersPermanent marker
?. [Non-destructive DNA extraction]
The first step prior to DNA extraction is to remove the insects from the sampling net, dry them, obtain biomass and then move on to DNA extraction. The general steps covered in this section are:Prepare labelsWeigh empty tubes with labelsRemove insects from the sampling netDry insect samplesWeigh the dry insectsSample digestionExtract digest and wash insects for sample preservation
?. [Prepare labels]
12 samples (11 samples + 1 blank) NB. A maximum of 12 samples can be extracted each day (per person).Make sure to make falcon tube labels for ‘ND extract’ and ‘ND extracted insects’, Microtube (Sarstedts 2 mL) labels for robot purification ‘QIASymphony aliquot’ and ‘QIASymphony purified DNA’ and four top labels for each sample (label size also fits Eppendorf tubes).Prepare labels for print. Label example:
?. [Weigh empty tubes with labels]
Designate and label tubes and weigh the empty tubes with labels standing on the lid – you will weigh them again after the insects have dried to get the dry weight of the sample
?. [Removal of insects from sample net (day 1)]
Clean the work area and laminar flow hood with1:10 diluted bleach and 70% ethanol. Sort insects in the laminar flow hood.Material and reagents needed (day 1)1:20 Bleach for cleaning 1 glass tub with spout for removal of insects from sampling net (can be substituted with plastic trays)70 & 96% EtOH   ForcepsVacuum filtration deviceFilters  Laminated mm paper for size sorting15/50 mL tubes for drying insects (label them with sample number and “10” (L = large) for size)Mask for respiratory protection during vacuum filtration 1 glass tub with spout for removal of insects from sampling net (can be substituted with plastic trays)70 & 96% EtOH   ForcepsVacuum filtration deviceFilters  Laminated mm paper for size sorting15/50 mL tubes for drying insects (label them with sample number and “10” (L = large) for size)Mask for respiratory protection during vacuum filtration ForcepsVacuum filtration deviceFilters  Laminated mm paper for size sorting15/50 mL tubes for drying insects (label them with sample number and “10” (L = large) for size)Mask for respiratory protection during vacuum filtration Laminated mm paper for size sorting15/50 mL tubes for drying insects (label them with sample number and “10” (L = large) for size)Mask for respiratory protection during vacuum filtration
?. [Removal of insects from sample net (day 1)]
Take a glass tub (with spout) and clean it with diluted bleach and 70% EtOH.
?. [Removal of insects from sample net (day 1)]
Pour the 96% EtOH from the sample into the tub and place the tub on the laminated mm paper
?. [Removal of insects from sample net (day 1)]
Take out a sample net and turn it inside out in the 96% EtOH in the glass tub. Most of the insects should let go when they come into contact with the ethanol
?. [Removal of insects from sample net (day 1)]
Try dipping the net up and down to make as many insects let go as possible. Add 70% ethanol if needed.
?. [Removal of insects from sample net (day 1)]
For the remaining insects, take a cleaned forceps (bleach + EtOH) and carefully remove them, releasing them into the 96% EtOH
?. [Removal of insects from sample net (day 1)]
When all the insects are free from the net, rinse the net and let it dry.
?. [Removal of insects from sample net (day 1)]
Take out any insects that are above 10 mm  – use the mm paper for picking them out. Make sure there is no smaller insects sticking to the larger insects. Place the larger insects in a separate falcon tube ( or  depending on how big they are). Each   tube should only contain max.   of insects and should be divided into two if they take up too much space.
15 mL
50 mL
50 mL
30 mL
?. [Removal of insects from sample net (day 1)]
Work with mask to avoid fumes from ethanol and bleach - alternatively work under fume extractor. The remaining insects in the ethanol are poured through a cleaned (bleach + EtOH)  vacuum filtration device and the insects that remain on the use-once filter.  (i.e. the small size fraction) is transferred to sample tubes ( or  depending on amount)
15 mL
50 mL
?. [Drying samples (predigestion) (day 1)]
Samples (with no lids) are placed in an oven at to dry. The fan should be off inside oven (if possible)! Lids are placed in a way to avoid contamination (e.g. within sheets of aluminium foil). Include a blank (an empty, open 15 mL tube) for each extraction day.Check the samples roughly every hour. EtOH leaking out of the insects can be removed with a p1000 to speed up the drying process and enable further processing the same day.
50 °C
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
When samples are dry (some samples may have ethanol residue almost impossible to get rid of) ad 'magic buffer' based on the original recipe: and later modified here: and lyse the samples. We use the modified version of the buffer.Material and reagents needed (day 1 or 2)70% EtOH for cleaning 1:20 Bleach for cleaning WeightWeighing boat Magic Buffer stock aliquot (see references for recipe)        DTT  Proteinase K P1000 pipette + tips1:20 Bleach for cleaning WeightWeighing boat Magic Buffer stock aliquot (see references for recipe)        DTT  Proteinase K P1000 pipette + tipsWeightWeighing boat Magic Buffer stock aliquot (see references for recipe)        DTT  Proteinase K P1000 pipette + tipsDTT  Proteinase K P1000 pipette + tipsProteinase K P1000 pipette + tips
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Clean the flow hood + pipette and pipette tip box prior to use
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Note the volume of the dried insects + falcon tube type (mL)
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Weigh the dry weight of the insects (biomass) in the falcon tube by placing the tube on its head in the center of the weight (mg).
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Calculate how much buffer is needed for all samples:The volume of insects in each tube +  buffer* (to ease the transfer of the supernatant), e.g. in sample a: of dried insects +  buffer =  for sample a*If there is less than  of insects in the tube, subtract the amount from the  total, e.g. insects –  buffer =  buffer should be poured on the sample.If there is  of 1-2 individuals of slim large insects (>=1 cm), add volume of insects +  buffer, e.g. in sample:  of 1 Syrphidae, +  = Add up the total amount of buffer need for all samples, and add 2 mL to the blank and prepare 1-2 extra mL to ensure you have enough buffer aliquot.
5 mL
3 mL
5 mL
8 mL
1 mL
5 mL
0.5 mL
5 mL
4.5 mL
1 mL
2 mL
0.5 mL
0.5 mL
2 mL
2.5 mL
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Take out an aliquot based on your calculation from the 500 mL stock in the fridge.
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Calculate how much DTT is needed for the reaction, e.g.: ( buffer aliquot · ) / 1000 =
40 mL
0.2468 g
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Calculate how much Proteinase K is needed for the reaction, e.g.:  buffer aliquot ·  =
40 mL
400 µl
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Add DTT and then Proteinase K and gently mix the reagents before adding it to the samples and make sure the DTT  completely dissolved. A good trick is to add DTT first, then buffer, and Proteinase K in the end. This way the DTT dissolves quickly. Add the buffer amount calculated for each sample, e.g. sample a = 8 mL of the buffer aliquot.
?. [Sample digestion (day 1 or 2)Non-destructive DNA extraction]
Place the samples in a rotator and put the rotator in an oven at  overnight. The rotator speed is the lowest setting (10).
50 °C
?. [Extracting digest (day 2 or 3)Non-destructive DNA extraction]
The supernatant is ready to be extracted from the insect samples. The insects should be preserved in 96% EtOH as bulk voucher samples.Material and reagents needed (day 2 or 3)70% EtOH for cleaning 1:20 Bleach for cleaning Large centrifugep1000 pipette + p1000 tips5 mL Eppendorf tubes for supernatant2 mL Sarstedt for aliquot of supernatant for purification 96% EtOH for conserving the insects1:20 Bleach for cleaning Large centrifugep1000 pipette + p1000 tips5 mL Eppendorf tubes for supernatant2 mL Sarstedt for aliquot of supernatant for purification 96% EtOH for conserving the insectsLarge centrifugep1000 pipette + p1000 tips5 mL Eppendorf tubes for supernatant2 mL Sarstedt for aliquot of supernatant for purification 96% EtOH for conserving the insects
?. [Extracting digest (day 2 or 3)Non-destructive DNA extraction]
Spin samples (only small size fraction) in the large centrifuge for about  . This will separate the insects somewhat from the supernatant to ease removal of the digest. If you have many small insects, then try to centrifuge the sample tube at 4000 g for 3 min  .
Centrifuge: 2000 34, 3 min
Centrifuge: 4000 34, 3 min
?. [Extracting digest (day 2 or 3)Non-destructive DNA extraction]
Extract digest with p1000 pipette (or p200/20 if many small insects get stuck in the tips) into 5 mL tubes (if there is more than 5 mL digest then discard the rest). This is a tedious process, but do make sure that no insects are transferred to the supernatant.
?. [Extracting digest (day 2 or 3)Non-destructive DNA extraction]
Fill sample tube with insects with 96% EtOH up to  and leave them until you finished extracting digest from all samples.
8 mL
?. [Extracting digest (day 2 or 3)Non-destructive DNA extraction]
Spin the samples (only small size fraction) in the large centrifuge for about   (if needed)
Centrifuge: 500 34, 1 min
?. [Extracting digest (day 2 or 3)Non-destructive DNA extraction]
Remove 96% EtOH from the samples and completely fill the tube with fresh 96% EtOH. If the EtOH looks clear then you can store the sample in the box. The samples that remain blurry should be flushed several times until the EtOH looks clear – this is occasionally the case for the samples with many small insects were it was difficult to remove the supernatant.
?. [Extracting digest (day 2 or 3)Non-destructive DNA extraction]
Vortex + spin the supernatant. Centrifuge settings .
Centrifuge: 2000 34, 1 min
?. [Extracting digest (day 2 or 3)Non-destructive DNA extraction]
Add  supernatant to sample tubes (2 mL Sarstedt tubes) for QIASymphony purification and prepare tubes with labels for the eluate.
230 µl
?. [DNA free laboratory]
Turn on the flow hood and clean the workbench during the startup of the flow hood
?. [DNA free laboratory]
Clean the pipette holder and the pipettes needed
?. [DNA free laboratory]
Clean the flow hood and place the pipettes, pipette holder and waste bin in the hood
?. [DNA free laboratory]
Clean and place a freeze rack in the hood and place PCR reagents (buffers, dNTPs, BSA) in the freeze rack to thaw
?. [DNA free laboratory]
Take 2 x 1.5 mL Eppendorf tubes – 1 for MM and 1 for molecular grade H2O. Label them.
?. [DNA free laboratory]
Fill the water tube with the water required for the PCR reaction and some extra for PCR negatives
?. [DNA free laboratory]
Mix the MM. Remember to add TagGold second last and H2O lastSLOWLY PUM UP AND DOWN WHEN ADDING THE H2O TO MIX THE BUFFER
?. [DNA free laboratory]
Take out the primers you need and place them in the first rack. The second rack is for the primer strips you are done with
?. [DNA free laboratory]
Take out as many PCR strips as you need, place them in the freeze rack and close the lids
?. [DNA free laboratory]
Label PCR strips. The PCR tubes should be labelled like this:
?. [DNA free laboratory]
Transfer MM to each PCR tube with the repeater. The amount of MM depends on how much DNA is added – check the recipe (e.g. 1 mL pipette tip, 19 µL MM, 32 x)
?. [DNA free laboratory]
Add primers to your PCR tubes. Use the multichannel pipette → place the first one on a tip and “rollover”” to get the rest of the tips. Make sure they all are fastened by rolling the pipette a bit from side to side while pressing down. First, add forward primers to all PCR tubes, then add the reverse primers
?. [DNA free laboratory]
Put the PCR strips and the rest of the H2O aliquot in a ziplock bag and store them in the fridge while you clean your workspace
?. [DNA free laboratory]
Take the ziplock bag and go to the DNA lab to add sample DNA
?. [DNA laboratory]
The PCR tubes have MM and tagged primers and are ready for DNA input in the DNA lab.Materials needed70% EtOH 1:20 Bleach Waste binMultichannel pipette10 µL pipette tipsPCR tubes with MM and tagged primersFreezing rack for PCR tube strips with MM and primers2 ng/µL DNA dilutions in PCR tube stripsRack for 2 ng/µL DNA dilutions PCR strips1:20 Bleach Waste binMultichannel pipette10 µL pipette tipsPCR tubes with MM and tagged primersFreezing rack for PCR tube strips with MM and primers2 ng/µL DNA dilutions in PCR tube stripsRack for 2 ng/µL DNA dilutions PCR stripsWaste binMultichannel pipette10 µL pipette tipsPCR tubes with MM and tagged primersFreezing rack for PCR tube strips with MM and primers2 ng/µL DNA dilutions in PCR tube stripsRack for 2 ng/µL DNA dilutions PCR strips
?. [DNA laboratory]
Turn on the flow hood and clean the workbench during the startup of the flow hood
?. [DNA laboratory]
Clean the pipette, tip box and racks
?. [DNA laboratory]
Clean the flow hood and place everything you need in the hood
?. [DNA laboratory]
Take out the DNA dilutions and allow them to thaw
?. [DNA laboratory]
When the DNA has thawed, vortex for ~ and spin them down for ~
?. [DNA laboratory]
Add DNA using the multichannel pipette and according to the spreadsheet recipe
?. [DNA laboratory]
Add some of the leftover PCR reaction H2O to the PCR blank(s)
?. [DNA laboratory]
Put the PCR strips and water back in the ziplock bag and store it in the fridge while you clean
?. [PCR laboratory]
Now the DNA has been added to your PCR reaction and you are ready to run the samples on the PCR machine.Place your samples in a PCR machine and adjust the settings according to the recipe on the spreadsheet. The run usually takes  - .
?. [PCR laboratory]
When there is ~. left, go to the gel area and prepare gels (2% agarose) corresponding to the number of samples you have
?. [PCR laboratory]
When the run is complete and your gels have solidified, prepare a sheet of parafilm
?. [PCR laboratory]
Use the multichannel pipette to transfer  gel red rows corresponding to the number of strips/samples you have
2 µl
?. [PCR laboratory]
Vortex and spin your samples
?. [PCR laboratory]
Put the gels in the gel chambers
?. [PCR laboratory]
Transfer  to the designated gel red droplets and mix the liquids by pumping up and down
5 µl
?. [PCR laboratory]
SET THE PIPETTE TO  AND PIPETTE EVERYTHING UP
7.3 µl
?. [PCR laboratory]
The first well and the last well should have ladder, so skip those
?. [PCR laboratory]
Starting from the second well, pipette the sample slowly into the bottom of the wellsDO NOT stab the gel! Gently feel your way to ease the tips into the wells. You can feel whether you are in the wells by slowly moving the pipette from side to side in the wells. You should feel the sidewalls of the wells if you are in the wells
?. [PCR laboratory]
When almost all liquid is out → lift the pipette and push almost all the way down → lift up the pipette slowly and suck in a bit before leaving the gel, to suck up any bubbles
?. [PCR laboratory]
Run the gel with settings: 130 V, 350 A, .
?. [PCR laboratory]
Now you can take pictures of the gels. Remember to put a USB stick in the USB slot and make sure the UV light is off.
?. [PCR laboratory]
Turn on the machine and go to live mode
?. [PCR laboratory]
Pour ddH2O on the surface and gently place plastic foil over it to avoid bubbles forming between the surface and the plastic foil.
?. [PCR laboratory]
Remove the gel from the gel chambers and pour off as much TBE buffer as possible. Make sure the gel does not slide
?. [PCR laboratory]
Gently push the gel onto the plastic foil and avoid bubbles forming under the gel
?. [PCR laboratory]
Close the door and turn on the UV-light
?. [PCR laboratory]
Adjust the setting so band appear visible, but no red colour is on the screen (red = too much light). Make sure to keep the settings the same when taking pictures of multiple gels
?. [PCR laboratory]
Save a picture on the USB stick by pressing “Save” – it takes some time
?. [PCR laboratory]
Print a picture by pressing “Print”. Make sure the photo machine is on. Label the photo immediately, so you know which picture is which and so you have a reference for the picture on your USB stick
?. [PCR laboratory]
Turn off the UV-light
?. [PCR laboratory]
Throw out the plastic foil and gel
?. [PCR laboratory]
Wipe the surface from water
?. [PCR laboratory]
Turn off the machine and take your USB stick
?. [PCR laboratory]
Immediately go to your computer and label your photo files from the USB stick correctly and update your gel results on the spreadsheet, see example Your PCR samples should be stored in the freezer for long time storage or in the fridge for short-time storage. DO NOT store samples in the fridge for more than a couple of days.
?. [Library build (Illumina TruSeq PCR-free)]
PCR products are now ready to be pooled into libraries for sequencing.All lab work must be carried out in the flow hood.Pool PCR products so your pools are equimolar.Make sure the starting DNA concentration > .Spin your buffers down before using them.Remember to take End Repair Mix 2 out of the freezer to thaw some time before beginning the library buildMake sure the starting DNA concentration >.Spin your buffers down before using them.Remember to take End Repair Mix 2 out of the freezer to thaw some time before beginning the library build
250 ng
?. [Pool samples]
Pool 96 samples with unique tag combinations, e.g.each tag only appears once on the forward and reverse primer - this should add up based on the PCR runs. This is to avoid tag jumps during sequencing. We pool the samples based on gel band strength, but you can use QuBit to check whether it corresponds to DNA concentration for a subset of samples before you begin. See example spreadsheet for pool setup
?. [Purify pools]
Purify using MinElute (fragments ). If the sample volume is over ~, then split the sample between two columns and combine them again after eluation (end volume = ).
10 µl
300 µl
40 µl
?. [Purify pools]
Remember to make aliquots for all buffers.
?. [Purify pools]
Aliquot the amount of buffer EB in eppendorf – put in a heatblock at .
37 °C
?. [Purify pools]
Add 5x volumes (rather more than less) PB buffer. Mix by pipetting up and down.  ABC1  Sample/Pool
    Sample/Pool volume (µl)
    PB added (µl)
  2   
     
     
  3   
     
     
  4   
     
     
  5   
     
     
  6   
     
     
  7
ABC1  Sample/Pool
    Sample/Pool volume (µl)
    PB added (µl)
  2   
     
     
  3   
     
     
  4   
     
     
  5   
     
     
  6   
     
     
  7
?. [Purify pools]
Spin per time on each column. You cannot load all sample at once, so repeat the step until all the PB has passed through the column.   + empty. If the sample does not pass at 6,000 x g, then increase the setting.
600 µl
Centrifuge: 6000 34, 1 min
?. [Purify pools]
Add  PE buffer to the column  .   + empty.
720 µl
Centrifuge: 10000 34, 1 min
?. [Purify pools]
Spin empty column for   to dry.
Centrifuge: 14000 34, 2 min
?. [Purify pools]
Put the filter in a new Eppendorf tube.
?. [Purify pools]
Add  EB to the center of the column. If the sample is split into two columns, then add 20 in each. Incubate at  for . Spin   to collect DNA. Turn tube. Repeat the spin.
25 µl
37 °C
Centrifuge: 14000 34, 1 min
?. [Purify pools]
Transfer to a new lo-bind tube and mark (combine the two replicates of each sample, if you split the columns).
?. [Purify pools]
Take out  of sample for the Qubit and 1 µL sample for the BA dilutions (conc.: ). Now the total volume for the sample is .
1 µl
38 µl
?. [Run Qubit (HS – high sensitivity) & Bioanalyzer (run later with other samples for the BA)]
ABC1  Sample
    Qubit Conc. (ng/µL)
    BA dilution (1:n)
  2   
   
     
     
  3   
   
     
     
  4   
   
     
     
  5   
   
     
     
  6   
     
     
  7   
     
     
  8  Library Blank
ABC1  Sample
    Qubit Conc. (ng/µL)
    BA dilution (1:n)
  2   
   
     
     
  3   
   
     
     
  4   
   
     
     
  5   
   
     
     
  6   
     
     
  7   
     
     
  8  Library Blank
?. [Illumnia TruSeq DNA PCR-free Library]
The following is for each library.
?. [Illumnia TruSeq DNA PCR-free Library]
Mix 40 µL End Repair Mix 2, 10 µL EB buffer (use the same as before or from the kit) and 50 µL purified PCR product in EB-buffer (adding EB buffer to ensue total reaction volume is 100 µL). Mix sample and EB buffer first, then add the End Repair Mix 2 (remember to spin down first). Pipet up and down to mix. Vortex and spin.  ABC1  Sample
    400/Qubit Conc. from previous table (µl)
  EBΔ (60 – value from the middle
  column)2   
   
     
     
  3   
   
     
     
  4   
   
     
     
  5   
   
     
     
  6   
     
     
  7   
     
     
  8  Library Blank
     
    60
  Put EB buffer back in heat-block.
ABC1  Sample
    400/Qubit Conc. from previous table (µl)
  EBΔ (60 – value from the middle
  column)2   
   
     
     
  3   
   
     
     
  4   
   
     
     
  5   
   
     
     
  6   
     
     
  7   
     
     
  8  Library Blank
     
    60
?. [Thermocycler]
Pre-heat lid to  for .Hold at
105 °C
30 °C
4 °C
?. [2nd MinElute purification]
Take new eppendorfs and add 5x volumes PB.
?. [2nd MinElute purification]
When the samples are done in the thermocycler, add the samples and mix by pipetting up and down.
?. [2nd MinElute purification]
Transfer to spin columns.
?. [2nd MinElute purification]
Spin  per time on column.   + empty.
600 µl
Centrifuge: 6000 34, 1 min
?. [2nd MinElute purification]
Add  PE buffer to column.   + empty.
750 µl
Centrifuge: 10000 34, 1 min
?. [2nd MinElute purification]
Spin empty column  to dry.
Centrifuge: 14000 34, 2 min
?. [2nd MinElute purification]
Put the filter in a new Eppendorf tube.
?. [2nd MinElute purification]
Add  EB to center of filter  . Incubate at for . Spin for   to collect DNA. Turn tube. Repeat the spin.
20 µl
37 °C
Centrifuge: 14000 34, 1 min
?. [2nd MinElute purification]
Transfer the libraries to PCR strip tubes.Freeze the samples in  if you do not proceed with the protocol at this time.
-20 °C
?. [2nd MinElute purification]
Mix  product from step 5.24 with  A-Tailing mix.
17.5 µl
12.5 µl
?. [Thermocycler]
Pre-heat lid to  for  for for Hold at Process next step immediately.
105 °C
30 °C
70 °C
4 °C
4 °C
?. [Add index]
Mix  product from step 5.27 with Resuspension buffer (EB). NB. .  DNA adapter Index (unique for each library) Ligation mix 2. NB. Add as the last thing (enzyme).Index primers  AB1  Library
    Index
  2   
     
  3   
     
  4   
     
  5   
     
  6   
     
  7   
     
  8  Library Blank
     
  37.5 µL total volumeSpin down.
30 µl
2.5 µl
4 Room temperature
2.5 µl
2.5 µl
AB1  Library
    Index
  2   
     
  3   
     
  4   
     
  5   
     
  6   
     
  7   
     
  8  Library Blank
?. [Thermocycler]
Pre-heat lid to  for .Hold at Spin down.
105 °C
30 °C
4 °C
?. [Thermocycler]
Place strips on cooler. Add  Stop Ligation Buffer and mix.Pipet up and down.Incubate  in cooler.Samples can be stored in the freezer at -20°C if you do not proceed with the protocol at this time.
5 µl
?. [Bead purification]
Take out 1 µL of sample for the Qubit and 1 µL sample for the BA dilutions. General protocol:
?. [Bead purification]
Check results on Qubit, and then Bioanalyzer.  ABC1  Sample
    Qubit Conc. (ng/µL)
    BA dilution (1:n)
  2   
   
     
     
  3   
   
     
     
  4   
   
     
     
  5   
   
     
     
  6   
     
     
  7   
     
     
  8  Library Blank
ABC1  Sample
    Qubit Conc. (ng/µL)
    BA dilution (1:n)
  2   
   
     
     
  3   
   
     
     
  4   
   
     
     
  5   
   
     
     
  6   
     
     
  7   
     
     
  8  Library Blank
?. [Bead purification]
Take out  beads from fridge and equilibrate the bead suspension to . (Take an aliquot, take the beads out from the fridge at least  before you take the aliquot!)
4 Room temperature
?. [Bead purification]
Make an (or reuse from library build) EB aliquot and place it in a heat block on
55 °C
?. [Bead purification]
Prepare aliquots fresh 80% ethanol from molecular grade ethanol (> per sample) and RNase free water (molecular grade).
400 µl
?. [Bead purification]
Measure the exact volume of PCR products and transfer to 1.5 ml tubes. Adjust volume to  with RNase free water.   ABC1  Sample
    Volume
    H2O added
  2   
   
     
     
  3   
   
     
     
  4   
   
     
     
  5   
   
     
     
  6   
     
     
  7   
     
     
  8  Library Blank
50 µl
ABC1  Sample
    Volume
    H2O added
  2   
   
     
     
  3   
   
     
     
  4   
   
     
     
  5   
   
     
     
  6   
     
     
  7   
     
     
  8  Library Blank
?. [Bead purification]
Vortex the beads thoroughly.
?. [Bead purification]
Add 0.8X bead volume (relative to PCR volume, 50 * 0.8 = 40 µl) to PCR product. Pipet gently but thoroughly at least 10 times until sample is completely homogenous.
?. [Bead purification]
Incubate at for .
55 Room temperature
?. [Bead purification]
Place on magnet for  or until solution has cleared. With the plate still on the magnet, remove the supernatant by pipetting. Save the supernatant. Take care not to disturb the beads, some liquid may remain in the tube.
?. [Bead purification]
While the tubes are still on the magnetic stand, add  of 80% ethanol using P200 without disturbing the beads and pipette slowly up and down three times.
200 µl
?. [Bead purification]
Incubate for  and then discard the supernatant without disturbing the beads.
?. [Bead purification]
Repeat step 5.40 and 5.41  . Remove all excess ethanol using P20.
?. [Bead purification]
Air-dry the tubes on the magnetic stand for - at  (not more!). The beads should appear crackled. Leftover ethanol will interfere with sequencing.
55 Room temperature
?. [Bead purification]
Remove the tubes from the magnet. Add  of  EB buffer to each sample and Mix by pipetting.
40 µl
55 °C
?. [Bead purification]
Resuspend the beads thoroughly by pipetting 10x.
?. [Bead purification]
Incubate at for at least .
55 Room temperature
?. [Bead purification]
Place the tubes back on magnet at for > until the beads clear from the solution.
55 Room temperature
?. [Bead purification]
Transfer the supernatant with the eluted library to new tubes. If some beads are still in the liquid, then you can use the pipet tip to push the beads up the side so the magnet can fixate them – you may need to do it a couple of times. Save the beads.
?. [Bead purification]
Measure DNA concentration on  library using Qubit.
1 µl
?. [Bead purification]
Dilute samples to approx.  for the BioAnalyser
?. [Bead purification]
Run samples on the BioAnalyser.
?. [Sequencing]
Example of setup for sequencing:  ABCDE1Lib #IndexPrimer start FPrimer start RSample Info2Library 34UDI0001fwhF2_001fwhR2n_001Carnet (IM18/19)3Library 35UDI0002insF2_001insR2n_001Carnet (IM18/19)4Library 36UDI0003fwhF2_001fwhR2n_009Carnet (IM18/19)5Library 37UDI0004insF2_001insR2n_009Carnet (IM18/19)6Library 38UDI0005fwhF2_001fwhR2n_017Carnet (IM19)7Library 39UDI0006insF2_001insR2n_017Carnet (IM19)8Library 40UDI0007fwhF2_001fwhR2n_025Carnet (IM19)9Library 41UDI0008insF2_001insR2n_025Carnet (IM19)10LibBlank5UDI0009
ABCDE1Lib #IndexPrimer start FPrimer start RSample Info2Library 34UDI0001fwhF2_001fwhR2n_001Carnet (IM18/19)3Library 35UDI0002insF2_001insR2n_001Carnet (IM18/19)4Library 36UDI0003fwhF2_001fwhR2n_009Carnet (IM18/19)5Library 37UDI0004insF2_001insR2n_009Carnet (IM18/19)6Library 38UDI0005fwhF2_001fwhR2n_017Carnet (IM19)7Library 39UDI0006insF2_001insR2n_017Carnet (IM19)8Library 40UDI0007fwhF2_001fwhR2n_025Carnet (IM19)9Library 41UDI0008insF2_001insR2n_025Carnet (IM19)10LibBlank5UDI0009