Instruction Manual

Vazyme Biotech Co., Ltd

Web: www.vazyme.com

Tel: 400-600-9335

Sales: Sales@vazyme.com

Support: Support@ vazyme.com

Address: Economic and Technological

Development Zone, Red Maple Park Building C1-2,

Kechuang Road, Nanjing, China

VAHTS Turbo End Repair/dA-Tailing Module

for Illumina®

Vazyme Cat #N201

Catalog

Components ..................................................................................................................................... 3

Storage Condition ............................................................................................................................ 3

Additional Required Reagents ........................................................................................................ 3

General Information ........................................................................................................................ 3

Introduction ............................................................................................................................. 3

Applicable Range ....................................................................................................................... 4

Quality Control .......................................................................................................................... 4

Detailed Operating Instructions ..................................................................................................... 5

Protocol ..................................................................................................................................... 5

Components

Components N201-01

(24 rxn)

N201-02

(96 rxn)

VAHTS Turbo End Prep Enzyme Mix 72 μl

288 μl

VAHTS Turbo End Prep Reaction Buffer (10 ×) 156 μl 624 μl

Storage Condition

All the components should be stored at -20°C for one year.

Additional Required Reagents

VAHTS Multiplex Oligos set 1/set 2 for Illumina® (Vazyme Cat#N302 or N303)

VAHTS Turbo Ligation Module for Illumina® (Vazyme Cat#N201-01)

VAHTS Super-Fidelity DNA Polymerase (Vazyme Cat#N206)

General Information

Introduction

VAHTS Turbo End Repair/dA-Tailing Module for Illumina® is a specially

developed kit of library preparation for high-throughput sequencing on the

Illumina platform. 5 ng – 1 μg of the DNA fragments ends can be repaired

effectively using this kit, with the phosphate groups of 5’ end and the dA-tailings

of 3’ end. Compared with conventional methods, VAHTS Turbo End

Repair/dA-Tailing Module for Illumina® Kit adopted one-step reaction process, to

skip multiple product purification steps, which reduces the minimum

requirements of the initial DNA template and shortens the experiment time

significantly. All the reagents passed strict quality control and functional

verification maximally guarantee the repeatability and stability of the library

construction.

Applicable Range

Applicable to the End Repair/ dA-Tailing from 5 ng- 1 μg fragmentation DNA

sample for high throughput sequencing on the Illumina platform.

Quality Control

VAHTS Turbo End Prep Enzyme Mix

SDS-PAGE Purity: SDS-PAGE analysis of each individual enzyme indicates over

95% purity. .

Endonuclease Activity: A 50 μl reaction system containing 10 μl of the enzyme

and 1 μg φX174 RF I DNA incubated at 37°C for 4 hours results in <10%

conversion to RF II detected by agarose gel electrophoresis.

Phosphatase Activity: Incubation of 1 µl of enzyme mix in protein

phosphatase assay buffer (1 M diethanolamine at pH 9.8 and 0.5 mM MgCl2 )

containing 2.5 mM p-nitrophenyl phosphate at 37°C for 4 hours yields no

detectable p-nitrophenylene anion as determined by spectrophotometric

analysis at 405 nm.

Functional activity detection: Add 1 μl of the enzyme and 0.5 μg of fragmental

DNA containing 5’ and 3’ jut-tailings to 1x End Prep Reaction Buffer, incubate for

20 minutes at 25°C, results in more than 95% of the DNA which is end repaired,

dA-tailed and phosphorylated.

VAHTS Turbo End Prep Reaction Buffer (10 ×)

16-Hour Incubation: A 50 μl reaction system containing 25 μl of VAHTS Turbo

End Prep Reaction Buffer and 1 μg of HindIII-λDNA incubated for 16 hours at

37°C results in no bands degraded detected by agarose gel electrophoresis. A 50

μl reaction system containing 25 μl of VAHTS Super-Fidelity 2x PCR Buffer and 1

μg of T3 DNA results in no bands degraded detected by agarose gel

electrophoresis.

Endonuclease Activity: A 50 μl reaction system containing 10 μl of the buffer

and 1 μg of φX174 RF I DNA incubated for 4 hours at 37°C results in less than 10%

conversion to RF II detected by agarose gel electrophoresis.

RNase Activity: Incubation of 40 ng FAM-RNA in 1×End Prep Reaction Buffer for

16 hours at 37°C results in no degradation bands detected by SDS - PAGE.

Phosphatase activity detection: Incubation of 1× End Prep Reaction Buffer

with 2.5 mM p-nitrophenylphosphate to Phosphatase Activity Detection Buffer at

37°C for 4 hours yields no characteristic absorption peak of nitrobenzene anion.

VAHTS Turbo End Repair/ dA-Tailing Module for Illumina®

All batches are tested and verified by the DNA library construction and being

sequenced on the Illumina platform.

Detailed Operating Instructions

Protocol

Experimental materials: 5 ng - 1 μg of fragmented DNA.

1. Prepare the following reaction mix in a sterilized PCR microtube:

VAHTS Turbo End Prep Enzyme Mix 3.0 μl

VAHTS Turbo End Prep Reaction Buffer (10 ×) 6.5 μl

Fragmented DNA x μl

ddH2O To 65.0 μl

2. Mix throughly by gently pipetting 10 times (do not shake), brief centrifuge to

collect the reaction liquid to the bottom of the tube.

3. Place the tube in a PCR instrument, and run the following reaction program:

Heating lip, 105°C

20 °C 30 min

65 °C 30 min

Hold at 4 °C

4. Directly use VAHTS Turbo Ligation Module (N201-01 or N201-02) to carry

out the adaptor ligation step.