(b) 4 cycles of:
l
98 ˚C for 80 s.
l
60 ˚C for 30 s.
l
72 ˚C for 30 s.
(c) 72 ˚C for 5 min.
4. Purify using KAPA pure beads kit as in Subheading 3.4, steps
4–15, using 1.8Â volumes of beads in step 5 instead of 1.2Â
volumes (see Note 9).
3.6 Testing
Library Size
1. Use 1 μL of sample (or 1 μL water, for the negative control)
from Subheading 3.5 as template for checking PCR. Combine
the following for each reaction (can be prepared as a master
mix) (25 μL final volume per reaction):
(a) 5 μL of 5Â Q5 buffer.
(b) 1.25 μL of 10 μM primer SSS401.
(c) 1.25 μL of 10 μM primer SSS402.
(d) 0.25 μL of Q5 Polymerase.
(e) 1 μL of DMSO.
(f) 1 μL of dNTP mix (mix is 10 mM each dNTP).
(g) 14.25 μL of molecular-grade water.
2. Perform PCR as follows, with reaction volume set to 100 μL
(see Note 7):
(a) 98 ˚C for 3 min.
(b) 25 cycles (see Note 10) of:
l
98 ˚C for 30 s.
l
60 ˚C for 20 s.
l
72 ˚C for 30 s.
(c) 72 ˚C for 5 min.
3. Run PCR reaction products on a 1.5% agarose gel to assess size
and abundance of products (see Notes 11 and 12).
3.7 Selection
and Purification
of Library Fragments
1. Clean gel box, tray, and comb thoroughly with soap and rinse
well with water (see Note 12).
2. Make a thick 1.5% (w/v) agarose gel containing ethidium
bromide.
3. Run all the PCR product from Subheading 3.5, step 3, dividing samples among multiple lanes as necessary (see Note 11).
4. Excise region of gel that contains a majority of product but
excludes primer-dimer.
5. Purify DNA using NEB Monarch DNA gel extraction kit as
Indicated in steps 6–16 below:
RNA 5
0
End Mapping
523
l
98 ˚C for 80 s.
l
60 ˚C for 30 s.
l
72 ˚C for 30 s.
(c) 72 ˚C for 5 min.
4. Purify using KAPA pure beads kit as in Subheading 3.4, steps
4–15, using 1.8Â volumes of beads in step 5 instead of 1.2Â
volumes (see Note 9).
3.6 Testing
Library Size
1. Use 1 μL of sample (or 1 μL water, for the negative control)
from Subheading 3.5 as template for checking PCR. Combine
the following for each reaction (can be prepared as a master
mix) (25 μL final volume per reaction):
(a) 5 μL of 5Â Q5 buffer.
(b) 1.25 μL of 10 μM primer SSS401.
(c) 1.25 μL of 10 μM primer SSS402.
(d) 0.25 μL of Q5 Polymerase.
(e) 1 μL of DMSO.
(f) 1 μL of dNTP mix (mix is 10 mM each dNTP).
(g) 14.25 μL of molecular-grade water.
2. Perform PCR as follows, with reaction volume set to 100 μL
(see Note 7):
(a) 98 ˚C for 3 min.
(b) 25 cycles (see Note 10) of:
l
98 ˚C for 30 s.
l
60 ˚C for 20 s.
l
72 ˚C for 30 s.
(c) 72 ˚C for 5 min.
3. Run PCR reaction products on a 1.5% agarose gel to assess size
and abundance of products (see Notes 11 and 12).
3.7 Selection
and Purification
of Library Fragments
1. Clean gel box, tray, and comb thoroughly with soap and rinse
well with water (see Note 12).
2. Make a thick 1.5% (w/v) agarose gel containing ethidium
bromide.
3. Run all the PCR product from Subheading 3.5, step 3, dividing samples among multiple lanes as necessary (see Note 11).
4. Excise region of gel that contains a majority of product but
excludes primer-dimer.
5. Purify DNA using NEB Monarch DNA gel extraction kit as
Indicated in steps 6–16 below:
RNA 5
0
End Mapping
523