2.4 PCR to Add
Full-Length Illumina
Adapter Sequences
and Amplify Libraries
1. Oligo SSS398: 5
0 AAT GAT ACG GCG ACC ACC GAG ATC
TAC ACT CTT TCC CTA CAC GAC GCT CTT C 3
0 , 10 μM
in water.
2. Reverse index primer: 5
0 CAA GCA GAA GAC GGC ATA
CGA GAT NNN NNN GTG ACT GGA GTT CAG ACG
TGT GCT 3
0 , 10 μM in water (index indicated in italics;
index sequences available from Illumina upon request).
3. Q5 polymerase with 5Â buffer.
4. Betaine: 5 M.
5. dNTP mix: 25 mM dATP, 25 mM dCTP, 25 mM dGTP, and
25 mM dTTP in water.
6. Molecular-grade water.
7. KAPA pure beads (Roche).
8. Magnetic separation stand that fits 1.5 mL microcentrifuge
tubes.
9. 80% ethanol, freshly prepared.
10. Thermocycler.
2.5 PCR to Amplify
Correctly Adapted
Molecules
1. Oligo SSS401: 5
0 AAT GAT ACG GCG ACC ACC GAG ATC
3
0 , 10 μM in water.
2. Oligo SSS402: 5
0 CAA GCA GAA GAC GGC ATA CGA GAT
3
0 , 10 μM in water.
3. Q5 polymerase with 5Â buffer.
4. Betaine: 5 M.
5. dNTP mix: 25 mM dATP, 25 mM dCTP, 25 mM dGTP, and
25 mM dTTP in water.
6. Molecular-grade water.
7. Thermocycler.
2.6 Testing
Library Size
1. Oligo SSS401: 5
0 AAT GAT ACG GCG ACC ACC GAG ATC
3
0 , 10 μM in water.
2. Oligo SSS402: 5
0 CAA GCA GAA GAC GGC ATA CGA GAT
3
0 , 10 μM in water.
3. Q5 polymerase with 5Â buffer.
4. Betaine: 5 M.
5. DMSO.
6. dNTP mix: 10 mM dATP, 10 mM dCTP, 10 mM dGTP, and
10 mM dTTP in water.
7. Molecular-grade water.
8. Agarose.
9. 1Â TAE buffer.
RNA 5
0
End Mapping
517