morgan@go2biotech.comNG303-TIANSeq Fast Ligation Module-200528
TIANSeq Fast Ligation Module
Cat.no. 4992354/4992355
Storage Conditions
TIANSeq Fast Ligation Module should be stored at -30~-15°C for one year. Avoid repeated freezing and thawing.
Product Description
The TIANSeq Fast Ligation module is a premixed enzyme module optimized for
illumina high-throughput sequencing platform. This module can be used to quickly
ligase adapters to the DNA fragments with dA-tailing at the 3’-end. Compared to
the conventional method, this module adopts the one-step reaction process, in
which the DNA fragments obtained from the TIANSeq Fragment/Repair/Tailing
Module (Cat# 4992350/4992351) or TIANSeq End Repair/dA-Tailing Module (Cat#
4992352/4992353) reaction could be used directly to the adapter ligation without
the need for purification.
This module eliminates the need for a multi-step magnetic beads purification step,
which makes the operation much easier and higher library construction efficiency
can be achieved.
Application: Quick ligation of DNA adapters to the DNA library fragments (for
example, the DNA fragments obtained from the TIANSeq Fragmen/Repair/Tailing
Module (Cat# 4992350/4992351) or TIANSeq End Repair/dA-Tailing Module (Cat#
4992352/4992353).
Sample input amount: 0.25 ng-1 µg DNA.
Other Recommended Reagents
- TIANSeq Fragment/Repair/Tailing Module(Cat# 4992350/4992351)
- TIANSeq End Repair/dA-Tailing Module(Cat# 4992352/4992353)
- TIANSeq Single-Indexed Adapter (illumina)(Cat# 4992641/4992642/4992378)
- TIANSeq Size Selection DNA Beads(Cat# 4992358/4992359/4992979)
Product Highlights
- The rapid ligation of adapters and DNA fragments can be performed without
the need for purification. - High ligation efficiency for the DNA-adapter ligation, even for low DNA input
amounts.
Precautions Please carefully read these precautions before using this kit.
- Attention should be paid in the operating process to avoid cross-contamination
between nucleic acid samples and products. - Please use RNase- or DNase-free pipette tips or EP tubes for the experiment.
- Before starting, wipe down work area with RNase and DNase cleaning reagents
such as RNase Away (Molecular BioProducts, Inc). Make sure there is no
contamination of RNase and DNase. - Before proceeding related operation, make sure the thermal cycler is calibrated
and in a stable state.
5.Please read the protocol carefully before the experiment. If test suspension is
needed or the downstream test is not needed to be carried out immediately,
the test products can be frozen and stored at -20°C and the subsequent test can
be planned accordingly.
Protocol:
- After the end repair/dA-tailing reaction, add Y μl adapter solution to the 50 μl
reaction mix, mix gently by pipetting and put on ice.
Notes: this kit does not contain the DNA adapter for sequencing. Please
refer to the usage conditions provided by the adapter supplier. TIANSeq
Single-Indexed Adapter (Illumina) (Cat# 4992641/ 4992642/ 4992378 ) is
recommended. To achieve higher ligation efficiency, we recommend the
molar ratio of the adapter to the DNA fragments in the reaction mix to
be between 10:1 to 200:1. For details, please refer to the TIANSeq SingleIndexed Adapter (Illumina) manual. - Prepare the reaction master mix according to the table below. Mix gently by
pipetting and then keep it on ice. - Add the prepared (50-Y) μl ligation master mix to the reaction solution prepared
in Step 1 to generate a 100 µl reaction mix, then gently mix by pipetting up and
down for 10 times. Incubate the ligation reaction at 20°C for 15 min.
Notes: if this step is performed using a thermal cycler, turn on the hot lid and
set the temperature ≤ 40°C. - It is recommended to use 1× (100 μl) TIANSeq Size Selection DNA Beads (Cat#
4992358/4992359/4992979) for the purification of ligation products. The steps
are as follows:
(1) Equilibrate magnetic beads at room temperature for 20 min.
(2) Vortex the magnetic beads to fully suspension. Add 100 μl of the thoroughly
vortexed the magnetic beads to the solution in Step 3, and mix well by
pipetting up and down for 10 times.
(3) Incubate the mix for 5 min at room temperature. Place the reaction tube on
the magnetic stand for 5 min. After the magnetic beads are completely
attached, carefully remove the supernatant.
(4) Place the tube on the magnetic stand and add 200-500 μl freshly prepared
80% ethanol (the ethanol should be just enough to immerse all the beads) to
the reaction tube, then gently pipette up and down for 3-5 times to wash the
magnetic beads (do not disturb the beads). Pellet the magnetic beads with a
magnetic stand for 30 sec and discard the supernatant.
(5) Repeat the washing in Step (4) once.
(6) Place the reaction tube containing magnetic beads on the magnetic stand,
open the lid and keep it at room temperature for 5-10 min until it is dried.
Note: do not over-dry magnetic beads, as this will cause a decrease in the
yield.
( 7) Remove the reaction tube from the magnetic stand, and thoroughly resuspend the
magnetic beads by adding 22.5 μl 10 mM Tris-HCl (pH 8.0) to the centrifuge
tube and gently mix by pipetting up and down for 10 times. Place the tube
at room temperature for 5 min, then put the reaction tube on the magnetic
stand for 5 min. When the magnetic beads are completely attached, transfer
about 20 μl of the supernatant to a new centrifuge tube for subsequent PCR
amplification.
(8)If size selection is needed, use 102.5 μl Nuclease-free ddH2O for beads
elution. Transfer 100 μl supernatant to a new centrifuge tube for subsequent
size selection.
Note:For size selection, please refer to the fragment size selection protocol
in TIANSeq DirectFast DNA Library Kit(illumina)(Cat# 4992259/4992260).
Alternatively, if library amplification is not intended for the ligation product,
add 12.5 μl of 10mM Tris-HCl (pH 8.0) to Step (6) for the elution of DNA, and
10 μl of the purified DNA can be transferred for subsequent application. If
not proceeding immediately, please keep samples stored at -20°C.
NG303-TIANSeq Fast Ligation Module-200528
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E-mail: maggie@go2biotech.com / morgan@go2biotech.com
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