McClean: Plasmid Prep Buffers: Difference between revisions

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(New page: Miniprep Buffers o Re-suspension Buffer (equivalent of Qiagen Buffer P1) � Tris·HCl – 50 mM � EDTA – 10 mM � RNase A – 100 μg/mL � HCl – final pH 8 (Note: Store RNase A ...)
 
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Miniprep Buffers
==Formulas for Qiagen Kit Buffers==
o Re-suspension Buffer (equivalent of Qiagen Buffer P1)
 
� Tris·HCl – 50 mM
Do not autoclave solutions containing ethanol, isopropanol or MOPS; use sterile filtration if necessary.
� EDTA – 10 mM
 
� RNase A – 100 μg/mL
Buffer AE  (elution buffer for genomic DNA preps)
� HCl – final pH 8
 
(Note: Store RNase A @ -20° C, aliquot buffer and add at time of use, do not
* 10 mM Tris-HCl
autoclave)
* 0.5 mM EDTA
(Note: Do not autoclave RNase A)
* pH 9.0
o Lysis Buffer (equivalent of Qiagen Buffer P2)
 
� NaOH – 200 mM
Buffer P1
� SDS – 1% (w/v)
 
(Note: Do not autoclave SDS, use sterile filter)
* 50 mM Tris-HCl pH 8.0
o Neutralization Buffer (equivalent of Qiagen Buffer N3)
* 10 mM EDTA
� Gu·HCl – 4.2 M
* 100 μg/ml RNaseA
� KOAc – 0.9 M
 
� HOAc – final pH 4.2
The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101).
o Column Wash/Binding Buffer (equivalent of Qiagen Buffer PB)
 
� Gu·HCl – 5.0 M
Buffer P2
� Isopropanol – 30 % (v/v)
* 200 mM NaOH
o Column Wash Buffer (equivalent of Qiagen Buffer PE)
* 1% SDS  (Sodium Dodecyl Sulfate)
� Tris·HCl – 10 mM
 
� Ethanol – 80% (v/v)
Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits)
� HCl – final pH 7.5
* 3.0 M potassium acetate pH 5.5
• Mini-column Recycling Buffer
 
o Equilibrium Buffer (equivalent of Qiagen Buffer QBT)
Buffer DP3 (for Qiagen Directprep 96-well miniprep)
� NaCl – 750 mM
* 3.0 M ammonium acetate pH 5.5
� MOPS – 50 mM
 
� Isopropanol – 15% (v/v)
Buffer N3
� Triton X-100 – 0.15% (v/v)
* 4.2 M Gu-HCl
(Note: Do not autoclave MOPS, use sterile filter)
* 0.9 M potassium acetate
• Protein Purification Buffer
* HOAc – final pH 4.2 (other sites say pH 4.8, but I've only tested 4.2 and it works)
o NID Extraction Buffer
 
� EDTA – 20-50 mM
Buffer PB
� Tris·HCl – 50 mM
* 5 M Gu-HCl
� NH₄Cl – 0.75 M
* 30% isopropanol
� Triton X-100 – 0.5% (v/v)
 
� Sucrose – 5.0% (w/v)
Buffer QG
� Lysozyme – 100 μg/mL
* 5.5 M guanidine thiocyanate (GuSCN)
� RNase A – 25 μg/mL
* 20 mM Tris HCl pH 6.6
� HCl – final pH 8
 
(Note: Store RNase A & Lysozyme @ -20° C, aliquot buffer and add at time of
Buffer PE
use, do not autoclave)
* 10 mM Tris-HCl pH 7.5
* 80% ethanol
* HCl- final pH 7.5
 
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
* Do not autoclave. Sterile filter
 
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)
* 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)
* 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
 
Buffer PNI (purification of oligonucleotides 17 nt and greater)
* 40% (v/v) 5 M guanidinium chloride
* 60% (v/v) isopropanol
  Concentration of guanidinium determined by drying down supplied concentrate and measuring mass. Concentration of isopropanol determined by calculation from the supplied user manual.
 
 
(Source: [http://methodsandreagents.pbwiki.com/], [[media:US6383393.pdf | US Patent 6,383,393]])
 
The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3

Revision as of 13:39, 22 May 2017

Formulas for 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 (Sodium Dodecyl Sulfate)

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
  • HOAc – final pH 4.2 (other sites say pH 4.8, but I've only tested 4.2 and it works)

Buffer PB

  • 5 M Gu-HCl
  • 30% isopropanol

Buffer QG

  • 5.5 M guanidine thiocyanate (GuSCN)
  • 20 mM Tris HCl pH 6.6

Buffer PE

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

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
  • Do not autoclave. Sterile filter

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)

  • 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)

  • 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

Buffer PNI (purification of oligonucleotides 17 nt and greater)

  • 40% (v/v) 5 M guanidinium chloride
  • 60% (v/v) isopropanol
 Concentration of guanidinium determined by drying down supplied concentrate and measuring mass. Concentration of isopropanol determined by calculation from the supplied user manual.


(Source: [1], US Patent 6,383,393)

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

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