Editing Plasmid buffers (section)

=== Buffer for Hangzhou silica Membrane column ===

Maxiprep:

'''Equilibration Buffer:''' 5~ 10%&nbsp; NaOH.

'''Neutralization Solution (N3)''': 2M Potassium Acetate, pH 5.5

'''12 ml Binding Solution''' ('''BS'''): 5% n-octyl-β-D-thioglucopyranoside, '''8M''' guanidine hydrochloride<br/>'''12 ml Wash Solution 1 (W1):''' 100 mM Tris Acetate, 4.5 M guanidine hydrochloride, 25% Isopropanol, pH 6.9<br/>'''12 ml Wash Solution 2''' '''(W2)''': 10 mM Tris-HCl, 10 mM NaCl, 80% Ethanol, pH 8.0

'''ETR:''' 20% Triton X-114 in isopropanol.

Other detegents:

1.5% BRIJ 35, 2.5% TWEEN 20, 1.0% TRITON X-100, 2.5% TRITON X-114

150 ml culture: 12 ml of P1, P2, N3; then add 8 ml BS; Load to column, wash with 12 ml BS, 12 ml W1, 12 ml W2. ELute with 3 ml TE (Spin).







'''Buffer AE''' (elution buffer for genomic DNA preps)

10 mM Tris-HCl<br/>0.5 mM EDTA<br/>pH 9.0

'''Buffer P1'''

50 mM Tris-HCl pH 8.0<br/>10 mM EDTA<br/>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<br/>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<br/>0.9 M potassium acetate<br/>pH 4.8

'''Buffer PB'''

5 M Gu-HCl<br/>30% isopropanol

'''Buffer QG'''

5.5 M guanidine thiocyanate (GuSCN)<br/>20 mM Tris HCl pH 6.6

'''Buffer PE'''

10 mM Tris-HCl pH 7.5<br/>80% ethanol

'''Buffer QX1 '''(for solution and binding of agarose gels)

7 M NaPO4<br/>10 mM NaAc<br/>pH 5.3

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

5 M GuHCl

'''Buffer QBT''' (equilibration buffer)

750 mM NaCl<br/>50 mM MOPS pH 7.0<br/>15% isopropanol<br/>0.15% triton X-100

'''Buffer QC''' (wash buffer)

1.0M NaCl<br/>50 mM MOPS pH 7.0<br/>15% isopropanol

'''Buffer QF '''(elution buffer)

1.25M NaCl<br/>50 mM Tris-HCl pH 8.5<br/>15% isopropanol

'''Buffer QN'''

1.6M NaCl<br/>50 mM MOPS pH 7.0<br/>15% isopropanol

'''Buffer FWB2'''

1M potassium acetate, pH 5.0

'''Buffer B1 '''(bacterial lysis buffer)

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

'''Buffer B2''' (bacterial lysis buffer)

3 M Gu-HCl<br/>20% Tween-20

'''Buffer C1''' (cell lysis buffer) (store at +4)

1.28 M sucrose<br/>40 mM Tris-HCl pH 7.5<br/>20 mM MgCl2<br/>4% Triton X-100

'''Buffer G2''' (digestion buffer)

800 mM GU-HCl<br/>30 mM Tris-HCl pH 8.0<br/>30 mM EDTA pH 8.0<br/>5% Tween-20<br/>5% Triton-X100

'''Buffer Y1''' (yeast lysis buffer) (store at +4)

1 M Sorbitol<br/>100 mM EDTA pH 8.0<br/>14 mM beta mercaptoethanol (added just before use)

'''Buffer PAA''' (PAGE gel elution of DNA)

500 mM NH4Ac<br/>100 mM MgAc2<br/>1 mM EDTA<br/>0.1% SDS



'''Column Regeneration'''

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.

The reuse protocol is:

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



1) When you are finished with the gel purification from Qiagen, place the used columns into the paired, used collection tubes.

2) Fill used column with 700 mL of 1 M HCl.

3) Store with cap closed in an airtight container for at least 24 hours (up to 30 days).

4) Once you have a bunch of columns and collection tubes in the 1 M HCl, to prepare a column for use:

Thoroughly rinse the columns and collection tubes in a large beaker of dH2O.

Shake/ wipe off excess water. Place each column into a collection tube. Fill each column with 700 uL sterile dH2O. Spin at maximum speed in a tabletop centrifuge for 1 minute. Discard the water.

Repeat the previous step a second time.

Equilibrate the column by filling it with 700 µL Buffer QBT. Spin at maximum speed in a tabletop centrifuge for 1 minute.

Discard the buffer. Spin again for 1 minute to remove excess QBT buffer from the column.

Put clean columns into a plastic bag. Rinse the collection tubes in a large beaker of dH2O.

Pour off water and allow tubes to air dry. Place into a plastic bag. The columns are now ready for use.

Note: After 9 rounds of regeneration, columns have shown optimal performance in a series of assays (Siddappa et al. 2007).

1M HCl

FW HCl = 36.47; 37% HCl = 370 g/L&nbsp;» 10 M

Note: for HCl, 1 Molar = 1 Normal

'''Qiagen Buffer QBT '''(Equilibration Buffer)

[750 mM NaCl, 50 mM MOPS (pH 7.0),15% ethanol, 0.15% Triton X-100]

Dissolve 43.83g NaCl, 10.46g MOPS (free acid) in 800 mL dH2O. Adjust pH to 7.0. Add 150 mL 100% ethanol and 15 mL 10% triton X-100 solution. Adjust volume to 1 liter.

Store at room temperature. Invitrogen Buffers<br/>==

'''RNase A''': 20 mg/mL in Resuspension Buffer (R3)<br/>'''Lysis Buffer (L7)''': 0.2 M NaOH 1% (w/v) SDS<br/>'''Precipitation Buffer (N3)''': 3.1 M Potassium acetate, pH 5.5<br/>'''Equilibration Buffer (EQ1)''': 0.1 M Sodium acetate, pH 5.0, 0.6 M NaCl, 0.15% (v/v); Triton X-100<br/>'''Wash Buffer (W8)''': 0.1 M Sodium acetate, pH 5.0, 825 mM NaCl<br/>'''Elution Buffer (E4)''': 100 mM Tris-HCl, pH 8.5, 1.25 M NaCl<br/>'''TE Buffer (TE)''': 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA