Team:TU Darmstadt/Materials/Quiagen buffers

From 2012.igem.org

Contents

Qiagen Kit Buffers

Do not autoclave solutions containing ethanol, isopropanol or MOPS; use sterile filtration if necessary.

Buffer AE (elution buffer for genomic DNA preps)

  • 10 mM Tris-HCl
  • 0.5 mM EDTA
  • pH 9.0

Buffer P1

  • 50 mM Tris-HCl pH 8.0
  • 10 mM EDTA
  • 100 μg/ml RNaseA

The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101).

Buffer P2

  • 200 mM NaOH
  • 1% SDS

Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits)

  • 3.0 M potassium acetate pH 5.5

Buffer DP3 (for Qiagen Directprep 96-well miniprep)

  • 3.0 M ammonium acetate pH 5.5

Buffer N3

  • 4.2 M Gu-HCl
  • 0.9 M potassium acetate
  • pH 4.8

Buffer PB

  • 5 M Gu-HCl
  • 30% isopropanol

Buffer PE

  • 10 mM Tris-HCl pH 7.5
  • 80% ethanol

Buffer QX1 (for solution and binding of agarose gels)

  • 7 M NaPO4
  • 10 mM NaAc
  • pH 5.3

Buffer QXB (for binding of large >3000 bp fragments to columns)

  • 5 M GuHCl

Buffer QBT (equilibration buffer)

  • 750 mM NaCl
  • 50 mM MOPS pH 7.0
  • 15% isopropanol
  • 0.15% triton X-100

Buffer QC (wash buffer)

  • 1.0M NaCl
  • 50 mM MOPS pH 7.0
  • 15% isopropanol

Buffer QF (elution buffer)

  • 1.25M NaCl
  • 50 mM Tris-HCl pH 8.5
  • 15% isopropanol

Buffer QN

  • 1.6M NaCl
  • 50 mM MOPS pH 7.0
  • 15% isopropanol

Buffer FWB2

  • 1M potassium acetate, pH 5.0

Buffer B1 (bacterial lysis buffer)

  • 50 mM Tris-HCl pH 8.0
  • 50 mM EDTA pH 8.0
  • 0.5% Tween-20
  • 0.5% Triton-X100
  • RNAse A 200 μg/l

Buffer B2 (bacterial lysis buffer)

  • 3 M Gu-HCl
  • 20% Tween-20

Buffer C1 (cell lysis buffer) (store at +4°C)

  • 1.28 M sucrose
  • 40 mM Tris-HCl pH 7.5
  • 20 mM MgCl2
  • 4% Triton X-100

Buffer G2 (digestion buffer)

  • 800 mM GU-HCl
  • 30 mM Tris-HCl pH 8.0
  • 30 mM EDTA pH 8.0
  • 5% Tween-20
  • 5% Triton-X100

Buffer Y1 (yeast lysis buffer) (store at +4°C)

  • 1 M Sorbitol
  • 100 mM EDTA pH 8.0
  • 14 mM beta mercaptoethanol (added just before use)

Buffer PAA (PAGE gel elution of DNA)

  • 500 mM NH4Ac
  • 100 mM MgAc2
  • 1 mM EDTA
  • 0.1% SDS

The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3

References

[1] http://methodsandreagents.pbwiki.com/

[2] [http://openwetware.org/images/1/1f/US6383393.pdf US Patent 6,383,393]

Recycling Qiagen Columns

The blue and purple Qiagen columns are identical in formulation. They are interchangeable and can be reused multiple times after treatment with hydrochloric acid, re-equilibration, and wash. Very likely this protocol can be used with other similar columns. Unused columns can be cheaply purchased in bulk from [http://www.epochbiolabs.com/minispin.asp?pageName=products Epoch Biolabs].

The reuse protocol is:

  • Save the collection tubes and columns after elution of DNA
  • Fill the column with 700 μl of 1 M HCl
  • Cap and store in an airtight container for at least 24 hours (less than a month)
  • Wash the columns and collection tubes in a large beaker of water
  • Assemble the column and collection tube and wash the column with 700 μl of DI water, discarding the water
  • Repeat
  • Fill the column with 700 μl of buffer QBT and spin down, discarding the buffer
  • Place the columns in an airtight plastic bag for storage
  • Wash the collection tubes, air dry, and store them for reuse

References

[1] http://www.pubget.com/paper/17373483

[2] http://www.bio.davidson.edu/courses/Molbio/Protocols/QIAQuick_recycle.html

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