McClean: Plasmid Prep Buffers

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 <-------This recipe has been verified in the McClean lab 5/1/2017

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

DO NOT AUTOCLAVE. Use a vacuum filter with 0.22μm pores (however, this has also been made without filtering so it might not be necessary). Store at 4°C. 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 <-------This recipe has been verified in the McClean lab

• 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

For 100 mL of PB buffer

• Pour < 60 mL of MilliQ water into a beaker.
• Add Gu-HCl = 47.78g.
• Stirring with hot plate turned on will help Gu-HCl dissolve faster
• Pour the solution into a 100 mL measuring cylinder
• Add more water to make a total of 70 mL solution.
• Add 30 mL of isopropanol. Stir the solution.
• Sterile filter the solution.

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

Contact

• Cameron J. Stewart 23 May 2017 (EDT):
• Megan N McClean 2017 (EDT)

or instead, discuss this protocol.