6. Incubate for 10 min at 25
C, then 50 min at 42 ˚C, and 15 min
at 70
C. Hold at 4
C.
7. Add 10 μL of each 1 N NaOH and 500 mM EDTA.
8. Incubate at 65 ˚C for 15 min.
9. Add 25 μL of 1 M Tris pH 7.5.
10. Purify with MinElute kit as in steps 11–17 below.
11. Add 600 μL of Buffer PB (Binding Buffer) to sample and
mix well.
12. Apply to MinElute column and centrifuge for 1 min at
>15,000 Â g.
13. Pour flow-through back over column, centrifuge again for
1 min at >15,000 Â g, and discard flow-through.
14. Apply 700 μL of Buffer PE (Wash Buffer) to column, centrifuge for 1 min at >15,000 Â g, and discard flow-through.
15. Repeat step 14 for a total of two washes.
16. Centrifuge for 1 min at >15,000 Â g to ensure membrane is
dry and then transfer column to a clean 1.5 mL tube.
17. Pipet 15 μL of molecular grade water onto membrane, centrifuge for 1 min at >15,000 Â g, and leave flow-through in tube.
3.4 PCR to Add
Full-Length Illumina
Adapter Sequences
and Amplify Libraries
Perform one reaction per sample, using SSS398 as a forward primer
with an indexed reverse primer of your choice.
1. Combine the following for each reaction (can be prepared as a
master mix) (50 μL final volume):
(a) 10 μL of 5Â Q5 buffer.
(b) 2.5 μL of 10 μM primer SSS398.
(c) 2.5 μL of 10 μM indexed reverse primer.
(d) 20 μL of 5 M betaine (see Note 6).
(e) 0.5 μL of Q5 polymerase.
(f) 0.4 μL of dNTP mix (mix is 25 mM each dNTP).
(g) 14.1 μL of cDNA (from step 17 in Subheading 3.3) or
molecular-grade water for no-template control.
2. Perform PCR as follows, with reaction volume set to 100 μL
(see Note 7):
(a) 98 ˚C for 3 min.
(b) 8 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.
RNA 5
0
End Mapping
521