TITLE: RADSeq protocol EFGL
AUTHORS: eaglefishgeneticslab

[DESCRIPTION]
Modified from protocol of Sean O’Rourke and Mike Miller published in:
 


[STEPS]
SECTION: Day 1 - Ligation
10. Ligate adapters onto digested DNA

 

SECTION: Day 1 - Ligation
10.1. Thaw BestRAD adaptors (prepared in steps 1-4) and reagents for Ligation Master Mix (listed above)

SECTION: Day 2 - Sonication
11. Prepping the Sonicator

 

SECTION: DNA quantification &lt;48 samples
7. Quantify DNA with Qubit

 



 

SECTION: Day 1 - Digestion
9. Digest the normalized DNA with a restriction enzyme


 

SECTION: Day 1 - Digestion
9.1. Thaw reagents to prep for master mix
Whichever restriction enzyme chosen for the project (PstI, SbfI, BamHI, or HindIII)
CutSmart Buffer 10X
Normalized DNA if it's been in the freezer
SECTION: Day 1 - Digestion
9.2. Add reagents from table below to a 1.5mL Lo-Bind tube to make a master mix. Add 5-10 to your sample size to account for pipette error. For example, if you have 20 samples, multiply the volumes per sample by 25 .

 
 
 

SECTION: Day 2 - Pool samples
12. 
 

SECTION: Day 2 - Sonicate, precipitate, ligate library
13. purifying pooled library

 

 

 


Using the table below, calculate the respective volumes required, or enter in number samples in "Pooling_calculator" tab in library prep spreadsheet to have volumes calculated
 
  
E.g. If N=48, then you need 3 tubes, the volume of pooled samples =256 µL, volume of AMPure XP beads = 256 µL, volume of 80% EtOH = 1.024 mL, volume of low TE = 70 µL
SECTION: Day 2 - Sonicate, precipitate, ligate library
13.1. Determine how many 1.5mL Lo-Bind tubes you need based on table above.
SECTION: Day 2 - Sonicate, precipitate, ligate library
14. Sonication

 

SECTION: Day 2 - Bead Selection
15. Prep buffers for bead binding in Step 17

 

SECTION: Day 2 - Bead Selection
15.1. Thaw 10X CutSmart Buffer at room temperature
SECTION: Day 2 - Bead Selection
16. Prep Dynabead M280 streptavidin magnetic beads

 

SECTION: Day 2 - binding DNA to beads
18. Liberate DNA from beads

 

SECTION: Day 2 - binding DNA to beads
18.1. Re-suspend beads in 100 µL of  1X CutSmart buffer from Step 15.3

Mix by pipetting

SECTION: Day 2 - binding DNA to beads
18.2. Place tube on the DynaMag Spin for 30 s 
SECTION: Day 2 - binding DNA to beads
19. Size select DNA fragments

 

19.1. Add 42 µL of AMPure XP beads to 42 µLDNA tube

Mix by pipetting
SECTION: Day 2 - Ligation
20. Ligating NEBNext adapters to DNA fragment

 

 

SECTION: Day 2 - Ligation
20.1. Thaw reagents from NEBNext End prep kit
Ultra II End Prep Enzyme Mix
Ultra II EP Buffer
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21. Size selection and cleanup of adaptor-ligated DNA

 


SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.1. Transfer all 86 µL solution from 96-well unskirted PCR plate to a new 1.5mL Lo-Bind tube

SECTION: Day 2 - PCR amp
None. PCR Amplification
Thaw the following reagents:
Ultra II Q5 Hot Start HiFi PCR Master Mix
Index Primer/i7 Primer (choose 1, need to be unique for each library)
Universal PCR Primer/i5 Primer

Add 25µl of Ultra II Q5 Hot Start HiFi PCR Master Mix to supernatant
Add 5µl of Index Primer/i7 Primer
Add 5µl of Universal PCR Primer/i5 Primer
Heat seal, gentle vortex, quick spin
SECTION: Day 2 - PCR amp
None. Run the thermalcycler program: 7 NEBNext PCR 11x (General -> RAD -> 7 NEBNext 11x)
Initial denaturation: 98°C for 30 seconds
Denaturation: 98°C for 10 seconds
Annealing/Extension: 65°C for 75 seconds, go to step 2 for 10 more times
Final Extension: 65°C for 5 minutes
Hold: 4°C

Transfer all 50μl PCR product to a new 1.5mL Lo-Bind tube (DO NOT stop here, stopping points are only after bead-size selection)
SECTION: Day 2 - Bead Clean-up
None. Cleanup of PCR Amplification
Add 45 µl of AMPure XP beads to PCR product in Lo-Bind tube, mix by pipetting or gentle vortex
Incubate at room temperature for 5 minutes, let sit in magnetic stand for 5 minutes
Remove and discard supernatant (~95 µl)
While still on magnetic stand, add 200 µl of fresh 80% Ethanol to beads and incubate for 30 seconds
Remove supernatant, and repeat wash step once
Leave tube in magnetic stand and allow any residual Ethanol to evaporate by letting stand for 5 minutes uncovered
Resuspend beads with 33 µl low TE, mix by pipetting/gently vortex, quick spin
Incubate for 2 minutes then place in magnetic stand for 5 minutes
Transfer 28 µl supernatant to a new 1.5mL Lo-Bind tube

This is now the completed RADseq library
Store library in -20°C if stopping for the day
SECTION: Day 3 - Qubit RAD library
None. Thaw library, and let it come to room temperature
Qubit library according to Qubit High Sensitivity Kit
Record qubit scores in library prep spreadsheet (Typical qubit range: 4-20ng/μl)
SECTION: Day 3 - E-gel
None. Run E-gel to get the approximate band size of RAD library
 

SECTION: Day 3 - E-gel
None. Thaw DNA ladder, gentle vortex, quick spin
SECTION: Day 3 - E-gel
None. 
Cut a strip of parafilm, use tape to adhere film to table if necessary
SECTION: Day 3 - E-gel
None. Unwrap e-gel, and remove comb
SECTION: Day 3 - E-gel
None. Select: "E-Gel 0.8-2%" program
Default time: 26 minutes
Start the run
Place the top camera back on machine
check gel at 20 minute mark

SECTION: Day 3 - E-gel
None. Tap on "View gel"
Dial up brightness, and take a picture
Export image to flash drive
Paste gel image in library prep spreadsheet
Determine band size of RAD library, and record that in the spreadsheet
SECTION: DNA quantification
6. Choose from the following options: 
Quantification of <48 samples
Quantification of >48 samples

SECTION: Annealing TOP/BOTTOM BestRAD adapters
1. Preparing BestRAD adapters (Skip section if BestRAD adapters have been previously annealed)

 
 
We typically make a 1µM working concentration of annealed adapters if the restriction enzyme we are using is a 6 base-pair cutter. We make a 50nM working concentration of annealed adapters if the restriction enzyme is an 8 base-pair cutter. A 6 bp cutter will cut more frequently so we need a higher concentration of it.

        
         
       

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
1.1. Making a 100µM stock of bottom and top adapters

Assuming the bottom and top adapters were ordered dry and at a 25 nmole scale and normalized to 10nmol, first re-hydrate to 100µM by adding 100 µL of nuclease-free water to each well with repeater pipette and combitip 

Heat seal the plate


 

SECTION: Day 1 - Ligation
10.2. gently vortex and spin down all reagents  

SECTION: Day 1 - Ligation
10.3. Make Ligation master mix in a 1.5mL Lo-Bind tube by combining reagents below. Multiply by the number of samples you have + 5-10 extra to account for pipette error.

   
SECTION: Day 1 - Ligation
10.4. Remove Digestion PCR plate from thermocycler (Step 9.9), quick spin   

SECTION: Day 1 - Digestion
9.3. Vortex and briefly spin down  

SECTION: Day 1 - Digestion
9.4. Label PCR plate "Digestion"

SECTION: Day 1 - Digestion
9.5. Using a p10 pipette or 0.1mL combitip, add 2 µLof master mix to each well

SECTION: Day 1 - Digestion
9.6. Using a multi-channel p10 pipette, add 10 µL of normalized DNA to corresponding wells

SECTION: Day 1 - Digestion
9.7. Heat seal PCR plate, vortex gently and quickly spin down.   

SECTION: Day 1 - Digestion
9.8. Run the following thermalcycler program (In our lab, the program is "1 RE-digest" and can be found using the menu options General -> RAD)
37 °C for 60 min
85 °Cfor 30 min                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            

CHECK THE PROGRAM TO MAKE SURE IT'S CORRECT
SECTION: Day 1 - Digestion
9.9. Set a timer for 75 min
Prep next step, about 15 min prior to end of thermalcycler program

 

SECTION: Day 1 - Ligation
10.5. With a multi-channel p10 pipette, add 2 µLof annealed adaptors to each well in Digestion PCR plate, corresponding to the same well setup in the "library prep" tab of BestRAD_library_prep_labData v1.2.xlsx. Discard tip after each use.                                                                                                                                            
SECTION: Day 1 - Ligation
10.6. Using a p10 pipette, add 2 µLof Ligation master mix to each well. Discard tip after each use.

SECTION: Day 1 - Ligation
10.7. Heat seal PCR plate, gently vortex, quick spin  

SECTION: Day 1 - Ligation
10.8.   
Run thermalcycler program: (In our lab, the program is called "2 Ligation" and can be found using the menu options : General -> RAD)
20 °C for 960 min
80 °Cfor 30 min


 


 




SECTION: qPCR
None. 
 
Manufacturer's protocol: https://www.neb.com/protocols/2015/05/14/nebnext-library-quant-kit-quick-protocol-e7630
SECTION: qPCR
None. Thaw the NEBNext Library Quant Kit 
SECTION: qPCR
None. Determine the number of serial dilutions needed from qubit score of RAD library

If Qubit scores are greater than 25ng/μl, use dilutions starting from 1:100,000 or 1:200,000
If any scores higher than 30ng/μl, recommended to have up to 1:2,000,000 dilution
SECTION: qPCR
None. Make sure Quant Master Mix in kit has been prepped. If not, 

add 500 µL of NEBNext Library Quant Primer Mix to the bottle of NEBNext Library Quant Master Mix (7.5 mL ). 
Mix by vortexing for 10 seconds. Write the date on the master mix bottle to indicate that primer mix has been added.
Add 100 µL (NEB#E7630L) ROX and vortex.

SECTION: qPCR
None. Make serial dilutions (are these in tubes? In triplicate?)
1:1000 dilution:         1 µL           RAD library                 + 999 µL1X Buffer
1:10,000 dilution:      10 µL       of 1:1000 dilution         + 90 µL 1X Buffer
1:100,000 dilution:    10 µL       of 1:10,000 dilution      + 90 µL 1X Buffer
1:200,000 dilution:    50 µL      of 1:100,000 dilution    + 50 µL 1X Buffer
1:500,000 dilution:    20 µL      of 1:100,000 dilution    + 80 µL 1X Buffer
1:1,000,000 dilution:  10 µL      of 1:100,000 dilution    + 90 µL1X Buffer
1:2,000,000 dilution:  50 µL     of 1:1,000,000 dilution + 50 µL 1X Buffer

After each dilution is made, vortex for 10 seconds, then quickly spin down, then make the next dilution.
SECTION: qPCR
None. Prepare 1X NEBNext Library Quant Dilution Buffer 

 150 µL10X Buffer + 1350 µL water
SECTION: qPCR
None. Using a 0.1mL combitip, dispense 8 µLof Quant Master Mix into wells in ABI MicroAmp Optical 7500 plate (More details of how many wells)

SECTION: qPCR
None. With a p10 single pipette, add 2 µL of standards (standards 2-5), (triplicate?) serial dilutions and NTC to corresponding wells. 

Pipette to side of wells to avoid bubbles
SECTION: qPCR
None. Seal with MicroAmp Optical Adhesive Film with a Kimwipe to avoid any smudges
SECTION: qPCR
None. DO NOT vortex plate, but do a quick spin
SECTION: qPCR
None. Load plate on ABI7500
In our lab, the program is "E7630 slow_v2"
Note that we use "Standard" mode, not fast
SECTION: qPCR
None. Export data.xls 
Enter results on Nebiocalculator to determine actual concentration for RAD library

Dilute RAD library to 4nM with 1X TE before proceeding with Illumina run.

LINK TO ILLUMINA PROTOCOL
SECTION: Day 3 - E-gel
None. Gentle vortex, quick spin RAD library and 6X loading dye
SECTION: Day 3 - E-gel
None. Remove top camera, and insert e-gel cartridge 
SECTION: Day 3 - E-gel
None. Load 20 µLof DNA ladder to lane 1, and 20 µL of RAD library to lane 2
SECTION: Day 3 - E-gel
None. Fill the remaining 10 empty lanes with20 µLof water
SECTION: Day 3 - E-gel
None. Close the lid, and power on the machine
SECTION: Day 3 - E-gel
None. Dispense a total of 12 water droplets: 1 at 5 µL, 1 at 12 µL, and 10 at 20 µL
SECTION: Day 3 - E-gel
None. Add 15 µLof DNA ladder to 5 µLwater droplet. Mix well by pipetting
SECTION: Day 3 - E-gel
None. Add5 µLof RAD library and 3 µLof 6X loading dye to 12 µL water droplet. Mix well by pipetting
SECTION: Day 2 - Sonication
11.1. Fetch ~2 L of de-ionized water from the Fish Health Lab. Take containers with you
  

SECTION: DNA quantification &lt;48 samples
7.1. Enter sample data info into “sampleData” tab of BestRAD_library_prep_labData v1.2.xlsx (attached in the DESCRIPTION tab of this protocol. It has example data for 20 samples.)
SECTION: DNA quantification &lt;48 samples
7.2. Qubit all samples with Qubit dsDNA HS assay kit. 
Follow the manufacturer's protocol  to make a working solution (1:200) of dye  https://tools.thermofisher.com/content/sfs/manuals/Qubit_dsDNA_HS_Assay_UG.pdf
SECTION: DNA quantification &lt;48 samples
7.3. Make sure you set the sample volume and the output to be read in ng/µl, not ng/mL

Take 2 readings per sample and enter them into columns F and G in "qubitData" tab in BestRAD_library_prep_labData v1.2.xlsx. The spreadsheet has a column that will take the average. The target is 10ng/µl. 
SECTION: DNA quantification &lt;48 samples
7.4. If DNA concentration is off the Qubit scale, 
perform a 1:5 dilution (1 µL of DNA, 4 µLof water) in a new PCR plate 
Re-Qubit diluted samples
SECTION: DNA quantification &lt;48 samples
7.5.  If DNA concentration is too low (less than 9ng/µl), reach out to lead biologist for replacement samples, or re-extract DNA then re-Qubit new samples
SECTION: DNA Normalization
8. Normalize DNA to 10ng/µl

 
The table in “Norm” tab of BestRAD_library_prep_labData v1.2.xlsx has been formulated to normalize all samples to 10ng/µl at a 30 µLfinal volume, and you’ll need 10 µLfor each sample in next step. 
SECTION: DNA Normalization
8.1. Label a new PCR plate: Normalized DNA for [insert RADseq project name], date, initials
SECTION: DNA Normalization
8.3. Pipette the amount of nuclease-free water indicated in column G of "Norm" tab to corresponding well position in new Normalization plate
SECTION: DNA Normalization
8.4. Heat seal plate, Mix by gently vortexing, then briefly spin the plate down  

 

SECTION: Day 2 - Sonication
11.3. Empty 1 L of de-ionized water into sonicator and start the machine. 
  

SECTION: Day 2 - Sonication
11.4. When the filter is fully saturated, and water cycle is complete, add more de-ionized water until the level is just below the top white spout.

  

SECTION: Day 2 - Sonication
11.5. Proceed to next step, and let machine cool to4 °C

SECTION: Day 2 - Sonication
11.2. Connect all corresponding tubes to the machine as labeled and screw on the filter   

SECTION: Day 2 - Pool samples
12.1. Pooling samples
Using a p200 single channel pipette, pool all 16 µL of each well from Digestion PCR plate into a new 1.5mL Lo-Bind tube
If library contains more than 93 samples, pool into a 2.0mL Lo-Bind tube instead

 


SECTION: Day 2 - Sonicate, precipitate, ligate library
13.4. Gentle vortex, quick spin.   

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.6. During the 10 min incubation, prep 80% ethanol in 5mL tube(s) or 15ml conical
 

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.7. Place tube(s) on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube(s) for 5 min

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.8. While the tube(s) are still on the magnet, using a p200 pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard supernatant 

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.9. While tube(s) are still on the magnet, add 2X total volume of 80% ethanol to the beads and incubate for 30 s. 

 


SECTION: Day 2 - Sonicate, precipitate, ligate library
13.10. Discard supernatant as in step 13.8

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.13. Leave tube(s) on magnet and allow any residual ethanol to evaporate by letting stand for 5 min uncovered

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.14. Take tube(s) off the magnet 
Add  low TE to each tube, refer to table at the beginning of this section for how much
Pipette up and down a few times to completely resuspend the bead pellet
SECTION: Day 2 - Sonicate, precipitate, ligate library
13.5. Incubate tube(s) at room temperature for 10 min

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.15. Incubate tubes(s) at room temperature for 5 min. 

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.17.  Using a p200 pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove the supernatant and put into a TPX tube.

Repeat for all library tubes on the magnet. Deposit all supernatants into same TPX tube
SECTION: Day 2 - Sonicate, precipitate, ligate library
13.12. Discard supernatant as in step 13.8
SECTION: Day 2 - Sonicate, precipitate, ligate library
13.16. Place tube(s) in magnetic stand for 5 min


 

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.18.  Pipette210 µL of water into another TPX tube

LABEL BOTH TPX TUBES TO AVOID MIXUP!!

 

SECTION: Day 2 - Sonicate, precipitate, ligate library
14.1. Sonicate library for 270 s at 20% capacity, and at 4 °C

SECTION: Day 2 - Sonicate, precipitate, ligate library
14.2. Remove tubes after sonication has completed

OK to sit in room temperature for next step. 
Discard TPX vial containing water
Turn off sonicator and coolant pump
 

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
2. Choose between the following options:
If using 6 base pair cutter (PstI, BamHI, HindIII), need to make 1µM dilution of BestRAD adapters
If using 8 base pair cutter (SbfI), need to make 50nM dilution of BestRAD adapters
SECTION: Annealing TOP/BOTTOM BestRAD adapters
3. Making a 1.0µM working concentration of COMBINED bottom and top adapters

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.1. Mix the 10µM adapter plates by gently vortexing, then briefly spin the plates down  

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.3. Using a multi-channel pipette, add2.5 µL  of the corresponding 10µM bottom adapters (the same well position as the top adapter plate) to the same well of the new plate. Discard tips.
SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.5. Using a repeat pipette and a 1mL combi-tip, add 45 µL  of duplex buffer to all wells. 

Heat seal the plate

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3. Making a 100nM concentration of SEPARATE adapters 

 



 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.1. Mix the 10µM adapter plates by gently vortexing, then briefly spin the plates down  

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.3. Use a repeat pipette and a 1mL combi-tip, add 99 µL  of nuclease-free water to each well of the new plate with an repeat pipette. 

Heat seal the plate
SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.4. Repeat for the bottom adapters, putting them into a separate, new plate. 

Heat seal the plate

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.5. Making a 50nM working concentration of COMBINED adapters


 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.6. Mix the 100nM adapter plates by gently vortexing, then briefly spin the plates down  

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.8. Using a multi-channel pipette, add 12.5 µL  of the corresponding 100nM bottom adapter (the same well position as the top adapter plate) from step 3.4 to the same well of the new plate. 
SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.9. Using a repeat pipette and a 1mL combi-tip, add 25 µL  of duplex buffer to all wells. 
Seal plate

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
4. Annealing adapters

Run the plate of COMBINED adapters (50 µL ) on a thermalcycler using the following program (In our lab, the program can be found using the menu options General -> RAD -> duplexAssay)

CHECK THE PROGRAM TO MAKE SURE IT'S CORRECT

95 °C for 2 min
Reduce temperature by 1 °C for 1 min
repeat until temperature is down to 4 °C





 


SECTION: Annealing TOP/BOTTOM BestRAD adapters
4. Continue with Step 4 under the 6bp cutter step case of the protocol




SECTION: DNA quantification &gt; 48 samples
7. Quantify DNA with plate reader

 


 
 
SECTION: Gen5 software
8. Prepare plate reader


 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
1.2. Making a 10uM stock of bottom and top adapters

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
1.3. Mix the 100µM adapter plates by gently vortexing, then briefly spin the plates down   
SECTION: Annealing TOP/BOTTOM BestRAD adapters
1.5. Using a repeater pipette and combitip, add 90 µL  of nuclease-free water to the same wells of the new plate. 

Heat seal the plate

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
1.6. Repeat for the bottom adapters from step 1.4. 

Heat seal the plate

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.2. Using a multi-channel pipette, add 2.5 µL  of the 10µM top adapters to a new plate. Discard tips.
SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.2. Use a p10 multi-channel pipette to transfer 1.0 µL  of each top adapter to a new plate. 
SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.7. Using a multi-channel pipette, add 12.5 µL  of the 100nM top adapter from step 3.3 to a new plate.
SECTION: DNA Normalization
8.2. Pipette the amount of DNA indicated in column F of "Norm" tab of BestRAD_library_prep_labData v1.2.xlsx from the DNA tray to corresponding well position in new Normalization plate

SECTION: Annealing TOP/BOTTOM BestRAD adapters
5. Check to see if there's sufficient stock made of 2X B+W buffer (needed for step 15, size selection) and stored in 50mL conical tube at room temperature. If not, make it
 

SECTION: Day 2 - Bead Selection
15.3. To a 1.5mL Lo-Bind tube add:
25 µL10X CutSmart Buffer
225 µL water
SECTION: Day 2 - Bead Selection
15.4. To a 1.5mL Lo-Bind tube add:

250 µL 2X B+W buffer
250 µLwater

SECTION: Day 2 - Bead Selection
15.5. To a 1.5mL Lo-Bind tube add:

175 µL 2X B+W buffer
175 µLwater
SECTION: Day 2 - Bead Selection
15.6. Place tube from step 15.5  in heat block set to 56 °C

 

SECTION: Annealing TOP/BOTTOM BestRAD adapters
3.4. Mix the duplex buffer by vortexing, then briefly spin down  
SECTION: Annealing TOP/BOTTOM BestRAD adapters
1.4. Use a p10 multi-channel pipette to transfer 10 µL  of each top adapter to a new plate. 
SECTION: Annealing TOP/BOTTOM BestRAD adapters
5.1. Add the following to a 50mL conical tube:
500 µL 1M Tris-HCl pH 7.5
100 µL0.5M EDTA pH 8.0
5.844 g NaCl
Add enough water to obtain 50 mL final volume

 

SECTION: Day 2 - Bead Selection
16.1. Vortex stock bottle of Dynabeads and transfer 20 µL to new 1.5mL Lo-Bind tube

 

SECTION: Day 2 - Bead Selection
16.2. Place tube on the DynaMag Spin for 10 s 

SECTION: Day 2 - Bead Selection
16.6. Place tube on the magnet for 30 s 

SECTION: Day 2 - Bead Selection
16.3. While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 20 µL supernatant 

SECTION: Day 2 - Bead Selection
16.4. Remove tube from magnet and add 100 µL 2X B+W buffer 
SECTION: Day 2 - Bead Selection
16.5.  Gently vortex for 30 s, then quick spin 
SECTION: Day 2 - Bead Selection
16.7. While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 100 µL supernatant 
SECTION: Day 2 - Bead Selection
16.8. Repeat steps 16.4 through 16.7
SECTION: Day 2 - Bead Selection
16.9. Take tube off magnet, re-suspend beads in 200 µL of 2X B+W buffer

Vortex

 

SECTION: Procedure Section
7. Launch Gen5 Software, and Task Manager will pop up. Click on Protocols in the left column. 
SECTION: Procedure Section
7.1. Choose a Plate Type
Select wells: Per step
Under Actions, select Read to add step in "Description" box

 
SECTION: Procedure Section
7.2. Double click on Read within "Description" box
Step Label: <default>
Filter Sets (we're only using 1), select "1"
Change both Excitation and Emission values to match the assay kit you're using, or choose the closest value available
Optics Position: Top (default)
Gain: 50
Leave remaining settings as default, Click OK.

 
SECTION: Plate Layout Section
8. Plate layout will pop up next, and the 96-well plate should have the following configuration as default. 
Columns 1 and 2 are set for Standards in duplicate
Remaining 80 wells are for samples

  

SECTION: Plate Layout Section
8.1. Right click on "STD (x16)", and select "Edit" to set up the DNA concentration standard curve range. 

  

SECTION: Plate Layout Section
8.2. Plate Layout ID = the abbreviation seen within the data table generated. 
Full Name = Full name of the abbreviation used for "Plate Layout ID"

Within "Conc. \Dil. values" tab, select "Concentrations" for Type, and enter in the appropriate Unit.
(No configuration needed in the "Colors" tab)

 Enter in the concentration values for each standard. The standard curve will be plotted using these values. The values are determined by how amount of each standard you add to each well. 
For example, 2µl of standard 3 (10ng/µl) = 20ng, 10µl of standard 7 (80ng/µl) = 800ng, etc.


 


Click OK to configure the next section

SECTION: Data Reduction Section
9. Recommended to include a Blank Transformation step, by setting Standard 1 (which should contain 0ng/μl) as the blank well

Add Blank step within the Transformations section
Standard Curve should already been included. If not, under Curve Analysis, add Standard Curve
Important! If Blank step is included, it must come before Standard Curve step.

  

SECTION: Data Reduction Section
9.1. Double click on Blank Transformation. 

Data In: Select the fluorescence reading
Blank Wells: STD 1
New Data Set Name: Blanked Fluorescence Data (Check the show box)
Formula: (not editable)

  Click OK

SECTION: Data Reduction Section
9.2. Double click on Standard Curve in Data Reduction table.

Curve Name: (leave as default)

Data In tab: Select "Generate Curve(s) from Current Plate" 
Well ID: STD
X Axis Data: <Concentrations/Dilutions>
Y Axis Data: Blanked Fluorescence Data

  

SECTION: Data Reduction Section
9.3. Curve Fit tab (leave all settings as default)

  

SECTION: Data Reduction Section
9.4. Data Out tab 

Data Set Name: DNA conc. [ng/ul]
Leave remaining settings as default

  Click OK

SECTION: Data Reduction Section
9.5. Save this .prt file to the Existing Protocol folder, and this template can be used for future DNA quantification within Gen5.
SECTION: Determining DNA concentration
9. Currently, the HTX plate reader is set to read DNA concentrations ranging from 0 to 200ng/μl (this is based on the amount of standards added). In the case where the raw DNA exceeds 200ng, you'll see a "?????" in the data table for DNA concentration.
SECTION: Day 2 - binding DNA to beads
17. Bind DNA to beads


 

SECTION: Gen5 software
8.1. Task Manager will pop up, and "Read Now" should be highlighted. If not, click on that. 

On the right panel, select "Existing Protocol..." folder. Select the .prt file to run.

  For future plate reads, the .prt files will be listed under Recent, and you can just click on that.

SECTION: Day 2 - binding DNA to beads
17.1. Transfer all 210 µL of sonicated library to freshly prepped 200 µLDynabeads tube from Step 16.9

SECTION: Day 2 - binding DNA to beads
17.2. Incubate for  20 min at room temperature while gently vortexing every 2 minutes for 10 seconds (e.g 20:00 minute mark, vortex till 19:50. At 18 minutes, vortex till 17:50, etc)
SECTION: Day 2 - binding DNA to beads
17.3.  Quick spin   then place tube on the DynaMag Spin for 30 s 
SECTION: Gen5 software
8.2. A new screen will load, and on the bottom right corner, it'll show a countdown of ~180 seconds for the bulb to warmup


 

SECTION: Day 2 - binding DNA to beads
17.4. While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 410 µL supernatant 
SECTION: Day 2 - binding DNA to beads
17.5. Take tube off magnet, re-suspend beads in 150 µL of 1X B+W buffer from Step 15.4
Mix by pipetting
SECTION: Gen5 software
8.3. Once bulb is warmed up, the screen will show that it is ready to load the Costar plate
Load the Costar plate on the plate reader, and click OK on the screen
Give it a couple seconds, and the machine will automatically close the plate loader.
SECTION: Day 2 - binding DNA to beads
17.6.  Place tube on the DynaMag Spin for 30 s 
SECTION: Gen5 software
8.4. The read will be completed in about a minute or two, and fluorescence readings for each well will populate on the screen.

Quick check if standards are loaded correctly, the readings should increase down the both columns.
SECTION: Day 2 - binding DNA to beads
17.7. While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 150 µL supernatant 
SECTION: Day 2 - binding DNA to beads
17.8. 
Repeat steps 17.5 - 17.7 2 more times



SECTION: Gen5 software
8.5. Once all wells have a read, click on "Statistics" tab, to view the data table. 

 

SECTION: Day 2 - binding DNA to beads
17.9.  Take tube off magnet, re-suspend beads in 150 µL of warm 1X B+W buffer from Step 15.6

Mix by pipetting


 

SECTION: Day 2 - binding DNA to beads
17.10. Place tube on the DynaMag Spin for 30 s 
SECTION: Day 2 - binding DNA to beads
17.11. While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 150 µL supernatant 
SECTION: Gen5 software
8.6. Click on the small green Excel looking icon beside the dropdown menu to have data table exported in Excel format. (additional configurations can be performed by clicking on "Edit Table", depending on your lab's needs)

 
SECTION: Day 2 - binding DNA to beads
17.12. Repeat steps 17.9 - 17.11 1 more time 
SECTION: Gen5 software
8.7. If you have more than 1 plate to read, save the Excel file first, and close out both Gen5 task page and Excel after. 
This is because the plate reader is set up to only read 1 plate at any time, and if you do not save and proceed with read of plate 2, it will overwrite all reads from plate 1 (this is not recoverable)

To exit the task page from Gen5, go to File > Close.
 

SECTION: Day 2 - binding DNA to beads
18.3. While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 300 µL supernatant 
SECTION: Gen5 software
8.8. Repeat steps 9 to 10.7 for additional plates
SECTION: Determining DNA concentration
10. Continue with Step 8 under the <48 samples step case of the protocol
SECTION: Determining DNA concentration
9.1. To find the actual concentration, refer to the standard curve's equation generated and make the appropriate calculation with the corresponding blanked fluorescence read of that sample. 

Each plate will have a slightly different equation, make sure to use the correct one.


 

SECTION: Day 2 - binding DNA to beads
18.4. Repeat steps 18.1 - 18.3. Note that this time around the volume of the supernatant to remove will be 100 µL

SECTION: Day 2 - binding DNA to beads
18.5. Take tube off magnet, re-suspend beads with bound DNA in 40 µL 1X CutSmart

Mix by pipetting

SECTION: Day 2 - binding DNA to beads
18.6. Transfer all 40 µL beads mix into well C3 of PCR plate 

 

SECTION: Day 2 - binding DNA to beads
18.7. Pipette  2 µLSbfI-HF (NEB R3642L) into the well

SECTION: Day 2 - Bead Selection
15.2. Check heat block to make sure it's on and set to56 °C
SECTION: Day 2 - binding DNA to beads
17.13. Take tube off magnet, re-suspend beads in 200 µL of 2X B+W buffer

Mix by pipetting


 

SECTION: Day 2 - binding DNA to beads
18.8. Use a p200 pipette set to 20μl to mix the solution 

DO NOT VORTEX or SPIN DOWN PLATE
SECTION: Day 2 - binding DNA to beads
18.9. Heat seal PCR plate
SECTION: Day 2 - binding DNA to beads
18.10. Run thermalcycler program: 3 37hold (General -> RAD -> 3 37hold)
37 °C for 60 min

SECTION: Day 2 - binding DNA to beads
18.11. After program has completed, give the plate a quick spin  

SECTION: Day 2 - binding DNA to beads
18.12. Place plate on magnet in plate format for 30 s 


 

SECTION: Day 2 - binding DNA to beads
18.13. transfer all 42 µL supernatant to new 1.5mL Lo-Bind tube

 


19.3. Place tube on the DynaMag Spin for 5 min 
19.2. Incubate 84 µL mixture at room temperature for 10 min

19.4. While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 84 µL supernatant 
19.5. While still on magnet, add 168 µL of  80% ethanol (made the same day) to beads and incubate for 30 s

19.6. While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 168 µL supernatant 
19.7. Repeat wash steps 19.5 - 19.6
19.8. Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for 5 min uncapped

19.12. Transfer 55 µLsupernatant to PCR plate (well C3 is preferred)

 

 

19.9. Resuspend beads with 55 µL low TE

Mix by pipetting
19.10. Incubate at room temperature for 5 min 

19.11. Place on DynaMag Spin for 5 min

 

SECTION: Day 2 - Ligation
20.4. Run thermalcycler program: 4 End Prep (General -> RAD -> End Prep)
20 °C for 30 min
65 °C for 30 min

Set a timer for 45 min
SECTION: Day 2 - Sonicate, precipitate, ligate library
13.2. Aliquot the pooled library into the tubes equally 
SECTION: Day 2 - Sonicate, precipitate, ligate library
13.3. Add an equal volume of AMPure XP beads to pooled library amount in a 1:1 ratio

SECTION: Day 2 - Sonicate, precipitate, ligate library
13.11. repeat step 13.9 one more time
SECTION: Day 2 - Ligation
20.2. Add 3 µL of Ultra II End Prep Enzyme Mix and 6.5 µL of Ultra II EP Buffer to the well of the tray with the  55 µL of DNA

Mix by pipetting (set p200 to 40µl)

SECTION: Day 2 - Ligation
20.3. Heat seal the plate and quick spin  

SECTION: Day 2 - Ligation
20.6. When timer goes off, thaw the following reagents for ligation:
Ultra Prep II Ligation master mix
NEBNext Adaptor
Ligation Enhancer
SECTION: Day 2 - Ligation
20.5. While program is running, drain sonicator. 
1. Disconnect the tube labeled “coolant supply” from the sonicator and remove the white nozzle end completely. 
2. Turn on the pump, and tip the sonicator backwards to drain as much water as possible, then turn pump off. 
3. Use paper towels to absorb all remaining water in sonicator
4. Leave lid open for a day or 2 to air dry. Unscrew the filter, empty out any remaining water and let it sit to air dry.
SECTION: Day 2 - Ligation
20.7. Dilute NEBNext adapter 1:10 in a new 1.5mL Lo-Bind tube
1 µL of NEBNext Adaptor + 9 µL 10mM Tris HCl pH 7.5


 

SECTION: Day 2 - Ligation
20.8. Make Ligation master mix in a 1.5mL Lo-Bind tube by combining reagents below. Multiply by the number of samples you have + 5-10 extra to account for pipette error.

 
 


SECTION: Day 2 - Ligation
20.10. Heat seal the plate, quick spin  
SECTION: Day 2 - Ligation
20.11. Run thermal cycler program: 5 Adaptor Ligation (General -> RAD -> 5 Adapter Ligation
20 °C for 15 min
SECTION: Day 2 - Ligation
20.12. After program has ended add 3 µL of NEBNext USER enzyme

Mix by pipetting as above


 

SECTION: Day 2 - Ligation
20.9. Add master mix to well of plate with DNA (the volume should be 83 µL)
Mix by pipetting (set p200 to 40µl and mix)
SECTION: Day 2 - Ligation
20.13. Heat seal the plate, quick spin   
SECTION: Day 2 - Ligation
20.14. Run thermalcycler program: 6 USER enzyme (General -> RAD -> 6 USER enzyme)
37 °C for 15 min

 

SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.3. Incubate at room temperature for 5 min 

SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.4. Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube for 5 min

SECTION: Day 2 - Ampure Size Selection and Clean-Up
None. Transfer 15µl supernatant to a 96-well unskirted PCR plate (well C3 is preferred)
If stopping is necessary, transfer supernatant to a new 1.5mL Lo-Bind tube and store in -20°C
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.2. Add 13.5 µL of nucelase-free water and 44 µL AMPure XP beads

mix by pipetting

SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.5. Transfer all 143.5 µL supernatant to new 1.5mL Lo-Bind tube

SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.6. Add 26 µL AMPure XP beads

mix by pipetting
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.9. Remove and discard supernatant (~170 µL)

SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.7. Incubate at room temperature for 5 min 
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.8. Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube for5 min

SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.10. While still on magnet, add 200 µL of fresh 80% ethanol to beads and incubate for 30 s

SECTION: Day 2 - Ampure Size Selection and Clean-Up
None. Remove supernatant, and repeat wash step once
Leave vial in magnetic stand and allow any residual Ethanol to evaporate by letting stand for 5 minutes uncovered
Resuspend beads with 17µl low TE, mix by pipetting/gently vortex, quick spin
Incubate for 2 minutes then place on magnetic stand for 5 minutes
SECTION: Day 2 - Ampure Size Selection and Clean-Up
None. 
SECTION: Day 2 - Ampure Size Selection and Clean-Up
None. 
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.11. Repeat 21.9 - 21.10
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.12. Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for 5 min uncovered
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.13. Take the tube off the magnet and re-suspend the beads in 17 µL low TE 

Mix by pipetting
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.16. Transfer 15 µL into a new unskirted PCR plate 
OR
If stopping for the day, transfer into a new 1.5mL Lo-Bind tube, store at -20 °C

 

SECTION: Day 2 - Ampure Size Selection and Clean-Up
22. PCR amplification
 

SECTION: Day 2 - Ampure Size Selection and Clean-Up
22.1. Thaw reagents from NEBNext Multiplex Oligo kit (orange box) labeled: “RADseq PCR Amp”
SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.14. Incubate for 2 min at room temperature

SECTION: Day 2 - Ampure Size Selection and Clean-Up
21.15. Put tube on DynaMag Spin for 5 min

SECTION: Day 2 - Ampure Size Selection and Clean-Up
22.2. Select an Index (i7) from the NEBNext Multiplex Oligo kit to use and record it in the "Final Library" tab in BestRAD_library_prep_labData v1.2.xlsx. 
 

SECTION: Day 2 - Ampure Size Selection and Clean-Up
22.3. Add the following reagents to the 15 µL of DNA:

  Total volume is 50 µL

SECTION: Day 2 - Ampure Size Selection and Clean-Up
22.4. Heat seal the plate, gently vortex, quick spin  

SECTION: Day 2 - Ampure Size Selection and Clean-Up
22.5. Run thermal cycler program: General -> RAD-> 7 NEBNext PCR 11x

Runs for about 28 min

  
 


SECTION: Day 2 - Ampure Size Selection and Clean-Up
22.6. Transfer 50 µL PCR product to new 1.5mL Lo-Bind tube


 


SECTION: Day 2 - Ampure Size Selection and Clean-Up
23. PCR purification

 

SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.1. Add 45 µL AMPure XP beads to the 50 µL of DNA from Step 22.6

mix by pipetting
SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.2. Incubate at room temperature for 5 min 
SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.3. Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube for5 min

SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.4. Remove and discard supernatant (~95 µL)

SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.5. While still on magnet, add 200 µL of fresh 80% ethanol to beads and incubate for 30 s

SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.6. Repeat 21.9 - 21.10
SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.7. Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for 5 min uncovered
SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.8. Take the tube off the magnet and re-suspend the beads in 33 µL low TE 

Mix by pipetting
SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.9. Incubate for 2 min at room temperature

SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.10. Put tube on DynaMag Spin for 5 min

SECTION: Day 2 - Ampure Size Selection and Clean-Up
23.11. Transfer 28 µL into a new 1.5mL Lo-Bind tube

Label the tube with species "Radseq", library number, date

Store at -20 °C if stopping for the day

 

SECTION: Day 3 - quantify library
24. Qubit Rad library

 

SECTION: Day 3 - quantify library
24.1. Thaw library to room temperature 
SECTION: Day 3 - quantify library
24.2. Make 1:200 diluted dye mix in a microcentrifuge tube
199 µL Qubit buffer
1 µL Qubit dye

SECTION: Day 3 - quantify library
24.3. Pipette 198 µL of diluted dye mix from Step 24.2 and 2 µL of the library into a qubit assay tube

Vortex thoroughly, quick spin   

SECTION: Day 3 - quantify library
24.4. Incubate at room temperature for 3 min

SECTION: Day 3 - quantify library
24.5. Put qubit assay tube in Qubit Fluorometer and take 2 readings per sample and enter them into BestRAD_library_prep_labData v1.1 spreadsheet in “Final Library” tab. The spreadsheet has a column that will take the average. The target is 4-20 ng/µl. 

 

 


SECTION: Day 3 - quantify library
25. E-gel of library 

 

SECTION: Day 3 - quantify library
25.1. Thaw 1 Kb ladder, loading dye, and RAD library if needed

Gentle vortex, quick spin   

SECTION: Day 3 - quantify library
25.2. Cut strip of parafilm, tape to benchtop if necessary
SECTION: Day 3 - quantify library
25.3. Onto parafilm, pipette 15 µLof 1 Kb ladder. Add5 µLof nuclease free water to the ladder. Mix by pipetting

SECTION: Day 3 - quantify library
25.4. Onto parafilm, pipette 5 µL of the RAD library. Add 3 µL of loading dye and 12 µL of nuclease free water to the library

RETURN LIBRARY TO FREEZER
SECTION: Day 3 - quantify library
25.5. Unwrap e-gel, remove comb, remove camera (top) from the Power Snap unit and load e-gel into machine
SECTION: Day 3 - quantify library
25.6. Pipette all 20 µL of diluted ladder from parafilm into lane 1 of e-gel

SECTION: Day 3 - quantify library
25.7. Pipette all 20 µL of diluted RAD library from parafilm into lane 2 of e-gel
SECTION: Day 3 - quantify library
25.8. Pipette 20 µL of water into remaining empty wells  of e-gel
SECTION: Day 3 - quantify library
25.9. Close lid, turn Power Snap on, select "E-gel 0.8-2%" program, push start, put camera back on Power Snap unit

Set timer for 15 min

 

SECTION: Day 3 - quantify library
25.10. When timer goes off,  tap "view gel" -> adjust brightness to max ->Take picture -> Tap "Export"-> insert thumb drive into usb port -> Tap "Export"
SECTION: Day 3 - quantify library
25.11. Paste e-gel picture in the BestRAD_library_prep_labData v1.1 spreadsheet under “Final Library” tab

Crop picture to include only lane1 and lane2

Use this image to determine approximate size of library and enter it into “Final Library” tab

  
 


SECTION: Day 3 - quantify library
26. qPCR library quantification

 

SECTION: Day 3 - quantify library
26.1. Thaw reagents in NEBNext library quantification kit and the RAD library 

Gently vortex the library, quick spin   
SECTION: Day 3 - quantify library
26.4. Make serial dilutions in 1.5 Lo-Bind tubes as follows:

 


 
1. 1:1000 dilution: 1 µL RAD library +999 µL1X Buffer
2. 1:10,000 dilution: 10 µL of 1:1000 dilution + 90 µL1X Buffer
3. 1:100,000 dilution:10 µL of 1:10,000 dilution + 90 µL 1X Buffer 
4. 1:200,000 dilution:50 µLof 1:100,000 dilution + 50 µL 1X Buffer 
5. 1:500,000 dilution: 20 µL of 1:100,000 dilution +80 µL 1X Buffer 
6. 1:1,000,000 dilution: 10 µL of 1:100,000 dilution + 90 µL1X Buffer 
7. 1:2,000,000 dilution: 50 µLof 1:1,000,000 dilution + 50 µL 1X Buffer 



SECTION: Day 3 - quantify library
26.3. Make sure the master mix in the qPCR kit has been prepped. Look for the "Prep checkbox". If it hasn't, follow instructions below

 
SECTION: Day 3 - quantify library
26.2. Check small bottle in fridge for 1X dilution of qPCR dilution buffer from NEBNext Library Quant kit. If low, make more:

150 µL10X Buffer 
1350 µL nuclease-free water 


SECTION: Day 3 - quantify library
26.5. Using a 0.1mL combitip, dispense 8 µLof Quant Master Mix to each well of a MicroAmp Optical 7500 Plate according to plate layout
 
SECTION: Day 3 - quantify library
26.6. Using a p10 pipette, add 2 µL of standards (use standards 2-5) from qPCR kit, library dilutions and no template control (NTC; 1X Buffer) to each well according to plate layout. Discard tip after each use.

 

SECTION: Day 3 - quantify library
26.7. Use MicroAmp Optical Adhesive Film to seal plate (this isn't heat sealed). Use a KimWipe to press down on the seal to avoid smudges

Quick spin    DO NOT VORTEX

Check underside of plate for dirt/lint, check wells for bubbles

SECTION: Day 3 - quantify library
26.8. Turn on 7500 qPCR system. Push to eject tray, load plate, push to close
SECTION: Day 3 - quantify library
26.9. Open 7500 v2.3 software
User name: GUEST
Click "Template" -> Open “E7630 slow_v2” (screen will be on “Plate setup”)
SECTION: Day 3 - quantify library
26.10. In left “Setup” column: Click on “Experiment Properties”
Enter in Experiment name (e.g. CarpRADseq_library1_09292022)
Leave everything else as is
SECTION: Day 3 - quantify library
26.11. In left “Setup” column: Click on “Plate Setup”
Click on “Assign Targets and Samples” tab
Highlight all wells that you’re not using à Right click and select “Clear”
SECTION: Day 3 - quantify library
26.12. Click on “Save as” and save to project folder on computer
Start Run (~60 min)

SECTION: Day 3 - quantify library
26.13. Export data.xls onto flash drive, and enter in results on NEB website to get the undiluted concentration of your RAD library
https://nebiocalculator.neb.com/#!/qPCRlibQnt

  
 


SECTION: Day 3 - quantify library
26.14. Dilute RAD library to 4nM using V1 = (C2*V2/V1) using 1X TE
 

Keep original and diluted libraries at -20 °C
 


Refer to "BESTRAD on NextSeq Protocol" for how to load the library

SECTION: DNA quantification &gt; 48 samples
7.1. Allow Quant-iT dsDNA BR assay kit to come to room temperature (at least30 min) 

Vortex and spin down all standards and dye.  

 

SECTION: DNA quantification &gt; 48 samples
7.2. Prep the working solution (1:200) in a 50mL reservoir 
For ~100 samples (add a couple of extra to account for pipette error):
100 µL of Component A from Quant-iT kit
20 mLof Component B from Quant-iT kit

Mix using a p1000 pipette.

 


 



SECTION: DNA quantification &gt; 48 samples
7.3. Using a multi-channel pipette, transfer 198 µL of working solution into each well of the Costar plate

 

 

SECTION: DNA quantification &gt; 48 samples
7.4. Add 2 µL of each standard from Quant-iT dsDNA BR assay kit in duplicate starting with the lowest concentration into columns 1 and 2. Discard tip after each use.

 


SECTION: DNA quantification &gt; 48 samples
7.5. Add 2 µL of samples' DNA to the remaining wells of the Costar plate, mix by pipetting and discard tips after each use. 
SECTION: DNA quantification &gt; 48 samples
7.6. Place Costar plate into a drawer (or a dark place), to incubate in the dark for 5 min