RAD protocol using 35 individuals per library.
1) Start with approximately 500 ng of genomic DNA from each of the DNA samples. Bring the volume
of each sample to 20 ul with H20 and transfer the samples into a 96-well plate.
2) Make SbfI digestion mix:
H20
NEBuffer 4 (10X)
SbfI-HF (NEB R3642L)
1X
2.15 ul
2.5 ul
0.25 ul
40X
86 ul
100 ul
10 ul
Rnase A(Qiagen-19101)
0.1 ul
4 ul
a) Add 5 ul to each well.
b) Incubate the plate at 37°C for 60 minutes.
c) Incubate the plate at 65°C for 20 minutes to inactivate the enzyme.
3) Add 1 ul of the appropriate barcoded SbfI P1 RAD adapter (50 nM) to each well in the sample plate.
(make sure adapter plate is thawed and spun down to eliminate contamination)
4) Make ligation mix:
H20
NEBuffer 4 (10X)
rATP (100 mM, Fermentas R0441)
T4 DNA Ligase (NEB M0202M)
1X
2.95 ul
0.5 ul
0.3 ul
0.25 ul
40X
118 ul
20 ul
12 ul
10 ul
a) Add 4 ul to each well.
b) Incubate the plate at 20°C for 60 minutes.
c) Incubate the plate at 65°C for 20 minutes to inactivate the enzyme.
5) Multiplex the samples that are to be sequenced together in the same library. Do this by combining 20
ul from each barcoded sample into a 1.5 mL Eppendorf tube. Divide into 6-0.5 ul tubes for
sonication (110 ul in each tube) (6 tubes total). Every individual that is multiplexed into the
same library must have a unique barcode. The remaining sample can be put in the freezer for
future use, if necessary.
6) Sonicate using the Bioruptor NGS on high with 15 seconds on and 90 seconds off for 5 cyles. Keep
sample tubes separate for the remaining protocol (Spin down samples and put on ice before
sonication)(Also, always balance out the Bioruptor with additional tubes at the same volume as your
sample tubes. There are 12 spaces in the machine.) (Save 2 ul for running on the gel).
7) Purify the samples (combine 3 tubes, 2 times for each library) with the Qiagen MinElute PCR
Purification Kit (28004) Do not exude 5 ug of Dna (Be sure to change tube after ethanol
throughput and extend spin for 3 minutes). Elute with 50 ul EB buffer (19086). Freeze one tube for future
use, if necessary.
8) Instead of gel extraction do an AMPure clean-up for size selection 1) Add 25 ul of AMPure XP beads
(Agencourt A63880) to the sample and let sit for 10 minutes. 2) Put on the magnet (Invitrogen DynaMag
12321D) and suck out the supernatant to put in another 1.5mL tube. Discard the beads. 3) Add another 25
ul of AMPure XP beads to the sample and complete the procedure as described in the AMPure protocol.
4) Elute in 20 ul EB. (Save 2 ul for running on the gel).
9) Blunt end repair the fragments by adding the following reagents to the sample in order:
Blunting buffer (10X)
dNTP mix (1 mM)
Blunting Enzyme Mix (NEB E1201L)
2.5 ul
2.5 ul
1.0 ul
• Incubate at 20°C for 60 minutes.
10) Purify the sample with using the AMPure XP protocol (use a 1:1 ratio of beads to sample volume). Elute
final step with 43 ul EB (save 2ul for running on the gel).
11) Add A-overhangs to the fragments by adding the following reagents to the sample in order:
NEBuffer 2 (10X)
dATP (10 mM)
Klenow Fragment (3' → 5' exo-) (NEB M0212L)
5.0 ul
1.0 ul
2.0 ul
• Incubate at 37°C for 60 minutes.
12) Purify the sample with using the AMPure XP protocol (use a 1:1 ratio of beads to sample volume). Elute
final step with 44 ul EB. (Save 2 ul for running on the gel).
13) Ligate the P2 adapter to the fragments by adding the following reagents to the sample in order:
NEBuffer 2 (10X)
P2 RAD adapter (10 uM)
rATP (100 mM, Fermentas R0441)
T4 DNA Ligase (NEB M0202M)
5.0 ul
1.0 ul
0.5 ul
0.5 ul
• Incubate at 20°C for 30 minutes.
14) Purify the sample with using the AMPure XP protocol (use a 1:1 ratio of beads to sample volume). Elute
final step with 50 ul EB. (Save 2 ul for running on the gel).
15) Make PCR mix:
H20
2X Phusion Master Mix (NEB F-531L)
P1 Adapter Primer (10 uM)
P2 Adapter Primer (10 uM)
Purified sample from step 14 (Store remaining at -20°C)
22.0 ul
50.0 ul
4.0 ul
4.0 ul
20.0 ul
• Place in thermal cycler and perform PCR. Cycling conditions: 98°C for 30 sec; 14X{98°C 10 sec,
65°C for 30 sec, 72°C for 30 sec}; 72°C for 5 minutes; Hold at 10°C.
*The volume of purified sample can be increased or decreased depending on the
concentration of your sample. If your starting DNA is low, then a higher amount of
purified sample can be used in your PCR step. Try not to increase the PCR cycles.
17) Purify the sample with using the AMPure XP protocol (use a 0.8:1 ratio of beads to PCR product). Elute
final step with 20 ul EB. (Save 2 ul for running on the gel).
18) Ran out 2ul of saved samples (from steps 6, 8, 10, 12, 14, and 17) on a 1% agarose gel to check
for PCR amplification as well as correct size selection.
18) Quantify the sample with an Invitrogen Qubit 2.0 Fluorometer (Q32866). Sample concentration may
vary depending on wear the sample is being sequenced, check with facility. Transfer sample to a nonstick 1.5ml tube. A bioanalyzer run as well as a qPCR are performed at the sequencing facility.