TF1 to TR1 amplicon is 5:8. Probe making is conducted in
50 μL reactions and mix the following reagents:
(a) PCR product mixture 1.3μg.
(b) 5μL10dNTP mix.
(c) 5μL10enzyme mix.- Mix and centrifuge briefly (15,000gfor 5 s).
- Incubate at 16C for 90 min.
- Add 5μL stop buffer.
- Add 1/10 volume 3 M sodium acetate and 2 volumes cold
( 20 C) ethanol to the reaction tube. Freeze at 70 C for
30 min. - Centrifuge at 15,000gfor 10 min. Carefully remove the
supernatant with a pipettor and dry the pellet. - Resuspend the pellet in 50μLH 2 O and precipitate the probe
with sodium acetate and ethanol as described above. - Resuspend the probe in TE buffer and store at 20 C.
3.2 Long DNA Library
Preparation
- LR-PCR products are mixed at a certain ratio the same with
step5in Subheading3.1. - Shear the mixture in a Focused-ultrasonicator M220 (Covaris)
by selecting the method DNA_2000bp_200_μL_Clear_micro-
TUBE for 12 min. The shear volume is 200μL. - End-repair reaction is carried out in 100μL reactions and mix
the following reagents:
(a) 130 ng sheared DNA.
(b) 20μL5end-repair buffer.
(c) 1μL end-repair enzyme. - Adaptor ligation is carried out in 100μL reactions and mix the
following reagents in Ion Plus Fragment Library Kit:
(a) 130 ng of sheared DNA.
(b) 1.6μL (Ion Xpress barcode adapter kits from 1 to 96).
(c) 10μL10ligase buffer.
(d) 2μL dNTP mix.
(e) 2μL DNA ligase.
(f) 8μL nick repair polymerase.
Incubate for 20 min at 25C in a thermal cycler followed by
72 C incubation for 5 min.- Select long DNA fragment by using 0.4 volume of Ampure
beads (i.e., 100μL sample of DNA gets 40μL of Ampure
beads).
36 Xing Chen et al.