Products / NGS Lib Prep Kit

StepWise DNA Lib Prep kit

Summary
Catalog No: RK20202
Input DNA amount: 500 pg-1000 ng
Size: 8 RXN / 24 RXN / 96 RXN
Sequencer: Illumina

To buy,select size,quantity

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Overview

The StepWise DNA library preparation kit is designed for illumina DNA sequencing library construction as little as 500 pg, up to 1000 ng. The kit can be used to perform PCR-free DNA library preparation, in which adaptors contain the complete sequence required for hybridization of the templates to the flowcell surface, and for annealing of sequencing primers. The minimal input for preparing PCR-free library is 100 ng of high quality genomic DNA, or 10 ng circulating cell-free DNA (cfDNA). PCR primers are included for users who wish to use self-selected DNA polymerase in the library amplification.
The entire workflow of StepWise DNA library preparation kit is shown as the below.

List of components


The Low EDTA TE buffer and PEG/NaCl solution can be stored at room temperature or 4°C. All other components should be stored at -20°C. The shelf life of all reagents is one year when stored properly.

Tube name8x RXN24x RXN96x RXN
End preparationEnd Prep buffer
End Prep enzymes
130 μl
25 μl
338 μl
65 μl
1.3 mL
250 μl
dA-PrepdA-Prep buffer
dA-Prep Enzymes
490 μl
10 μl
1274 μl
26 μl
4.9 mL
100 μl
Adaptor ligationLigase MM
Ligase mix
255 μl
20 μl
663 μl
52 μl
2.55 mL
200 μl
Amplification2x PCR master mix
PCR primers
Low-EDTA TE
250 μl
15 μl
1 mL
650 μl
39 μl
3 mL
2.5 mL
150 μl
10 mL
OthersPEG/NaCl solution1 mL3 mL10 mL

Additional Materials required


  • Singleplex or multiplex adaptors for illumina.
  • 80% ethanol (freshly prepared, room temperature).
  • PCR tubes or plates.
  • Magnetic stand.
  • Thermocycler.
  • Agencourt AMPure XP bead (room temperature).
  • Pipettes/multichannel pipettes (2.5, 10, 20, 200, 1000 μl).
  • Minicentrifugator.
  • Vortex.

General Consideration


01. Please you use high quality DNA as possible as you can. Heavily nicked or damaged DNA may cause library preparation failure. You can use 260 nm/280 nm ratios (1.8~2.0) to check DNA purity. You also can use fluorescent method to perform double-stranded DNA quantification. Contaminating RNA, nucleotides and single-stranded DNA may affect the amount of usable DNA in a sample preparation.

02. Please mix by gently pipetting the reaction mix up and down.

Protocol


After adaptor ligation, clean up step is designed to remove low molecular weight materials. Size selection step is also designed for users who want to perform size selection for their libraries. If your input DNA is less than 50 ng, size selection is not recommended.

The kit also can be used to perform PCR-free DNA library preparation, in which adaptors contain the complete sequence required for hybridization of the templates to the flowcell surface, and for annealing of sequencing primers.You also can use customized or other commercial PCR-free Y-shape adaptors for illumina sequencing plateform.

In the amplification step, you also can use yourself-selected DNA polymerase, and PCR primers are included in this kit.

Oligonucleotide Sequences


Adaptor:


Primer1:  5’-AATGATACGGCGACCACCGAG

Primer2:  5’-CAAGCAGAAGACGGCATACGAG

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