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gDNA Fragmentation Kit (AluI/RsaI-based)

Cat. No. Amount Price (EUR) Buy / Note
APP-005 20 reactions x 50 μl 126,88 Add to Basket/Quote Add to Notepad

For research use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Description:
gDNA Fragmentation Kit (AluI/RsaI-based) contains all reagents for sequence-specific enzymatic fragmentation of genomic DNA (gDNA) either purified or directly derived from WGA reactions (see #APP-004).
Both AluI and RsaI possess a four base-pair recognitions sequence (AluI: AG↓CT, RsaI: GT↓AC).
The resulting fragmented gDNA can subsequently be used for downstream applications such as preparation of hybridization probes via Klenow fragment-based DNA labeling.[1-3]

Content:
AluI
2x 600 units (10 U/μl) in 10 mM Tris-HCl pH 7.4, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA and 50 % [v/v] glycerol

RsaI (10 U/μl)
1000 units (10 U/μl) in 10 mM Tris-HCl pH 7.4, 50 mM KCl, 0.1 mM EDTA, 200 μg/ml BSA and 50 % [v/v] glycerol

10x Universal Buffer (UB)
1 ml

BSA
1 ml (10 mg/ml) in PCR-grade H2O

DTT
20 μl, 100 mM in PCR-grade H2O

PCR-grade H2O
1.2 ml


General protocol for gDNA fragmentation
The protocol below is intended as a general guideline to generate gDNA fragments ranging from 250-1000 bp however, individual optimization might be required e.g. in terms of template amount, incubation time or AluI/RsaI concentration.

Reaction set-up

  • Transfer 1-4 μg of purified genomic DNA (gDNA) or up to 18 μl WGA reaction mix (e.g derived from # APP-004) to a prechilled DNAse/RNAse free reaction tube (e.g. 1.5 ml Eppendorf tube).
  • Add all components described below on ice.


ComponentFinal conc./amount1 reaction
Genomic DNA1-4 μg purified gDNA or up to 18 μl WGA MixX μl
10x Universal Buffer1x5 μl
AluI (10U/μl)50U5 μl
RsaI (10U/μl50U5 μl
BSA (10 mg/ml)5 μg0.5 μl
DTT (100 mM)1 mM0.5 μl
PCR-grade H2O-ad 50 μl

  • Vortex and spin-down briefly.
  • Incubate for 2h at 37°C (e.g. Thermomixer, 300 rpm).
  • Inactivate the enzyme for 10 min at 65°C.
  • Spin-down briefly
  • Proceed with down-stream application (e.g. purification of gDNA fragments (#PP-201, following High Yield Sample preparation protocol 1b) and subsequent Klenow fragment-based labeling). Alternatively, store fragmented gDNA at -20°C until usage and avoid repeated freeze-thaw-cycles.


1.2 Evaluation of gDNA fragmentation

  • gDNA fragmentation can be determined via agarose gel electrophoresis (2-5 μl reaction mix, 2.4% agarose gel (TAE Buffer)). The average product length will range from 250 bp and 1000 bp in most cases.
  • Adjust reaction parameter according to your desired product length (template amount, restriction enzyme amount, incubation time).

Selected References:
[1] Hostetter et al. (2010) Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples. Nucleic Acid Res. 38 (2): e9.
[2] Kawachi et al. (2013) Deletion polymorphism at chromosome 3q26.1 and oral squamous cell carcinoma. International Journal of Oncology 42:384.
[3] Uda et al. (2007) Comparison of Whole Genome Amplification Methods for Detection of Pathogenic Bacterial Genomic DNA Using Microarray. Jpn. J. Infect. Dis. 60:355.