Editing Plasmid buffers (section)

== Silica membrane, low endotoxin ==

'''Binding Solution''': 5% n-octyl-β-D-thioglucopyranoside, 8M guanidine hydrochloride.

Add 9 ml to 36 ml clear lysate (12 ml P1, 12 ml P2, 12 ml P3)--> Final conc is 1.6 M of guanidine hydrochloride, alternatively, use N3 containing 4.8 M guanidine hydrochloride. If N3 is used, wash column with Low-endotoxin Wash before washing with PB and PE.



'''PB''': 100 mM Tris Acetate, 4.5 M guanidine hydrochloride, 25% Isopropanol, pH 6.9<br/>'''PE''': 10 mM Tris-HCl, 10 mM NaCl, 80% Ethanol, pH 8.0

'''Low-endotoxin Wash''': 1% n-octyl-β-D-Solution thioglucopyranoside, 1.6 M guanidine hydrochloride







'''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 ug/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

'''Buffer ENDO-1'''

50 mM MOPS or HEPES (pH 7.00 with NaOH)<br/>750 mM sodium chloride<br/>10% (w/v) Triton X-114<br/>20% (v/v) isopropanol

<br/>'''Buffer W5-ENDO'''

100 mM sodium acetate (pH 5.0 with acetic acid)<br/>750 mM sodium chloride<br/>1% (w/v) Triton X-114



'''Buffer W7-ENDO, Qiagen ER buffer'''

50 mM MOPS (pH 7.0 with NaOH)<br/>750 mM sodium chloride<br/>10% (w/v) Triton X-114 or 10% (w/v) Triton X-100 or 20% NP 40

10% ethanol

Add to cleared lysate at 10:1 ratio, e.g., add 3 ml to 30 ml cleared lysate.

<br/>'''Stock Solutions:'''<br/>'''0.5 M MOPS pH 7.0'''<br/>209.27 g MOPS (use the free acid form that USB sells)<br/>~750 ml dH20<br/>pH to 7.0 with 10 N NaOH<br/>make up to 2 liters with dH20<br/><br/>'''3M NaCl'''<br/>350.6 g NaCl<br/>make up to 2 liters with dH20

'''1 M Tris pH8.5'''<br/>121.1 g Tris base<br/>~750 ml dH20<br/>pH to 8.5 with concentrated HCl (takes ~20 ml)



'''PCR purification buffer'''

'''QXB''': 5 M GHCl



'''PB''': 5 M GuHCl, 30% isopropanol<br/>20mM Tris-HCl pH 6.6 (25oC)

'''Buffer QG 1&nbsp;'''(neutralization and binding buffer)<br/>5.5M guanidine thiocyanate (GuSCN)<br/>20mM Tris-HCl<br/>pH 6.6 (25oC)



'''Recipe 2'''<br/>90.8 g NaI<br/>1.5 g Na2SO3<br/>100 ml of water.<br/>Filter through 0.45 um filter,<br/>then add another 0.5 g Na2SO3.<br/>Store in the coldroom in a dark bottle.

'''Recipe 3'''<br/>6M Guanidine thiocyanate, Molecular Weight: 118.16, 71 g<br/>50mM Tris-HCl pH 7.5<br/>20mM EDTA pH 8.0

water to 100 ml



'''Recipe 4'''<br/>60% Guanidine thiocyanate (5.36M), 60 g<br/>140mM MES (2-[N-Morpholino] ethanesulfonic acid)<br/>0.006% Phenol Red

H2O to 100 ml

0.006% Phenol Red Use 3 Volume of buffer to 1 gel volume.

Reference: <a href="[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750]" alt="[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750]" title="[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750]">[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750 http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007750]&lt;/a&gt; Phenol Red is only a pH indicator. It changes color from yellow to red over the pH range 6.8 to 8.4.

7/3/2013



&lt;img src="/eln/images/3/39/Gel_extraction_buffer_comparison.JPG" _fck_mw_filename="Gel extraction buffer comparison.JPG" _fck_mw_origimgwidth="159" _fck_mw_origimgheight="289" alt="RTENOTITLE" title="RTENOTITLE" style="vertical-align:middle;" /&gt;<br/>H: Homemade gel extraction buffer

Q: Qiagen gel extraction buffer QG.

<br/>'''Genomic DNA buffer'''

'''Chaotreopic Solution''': 4 M GTC, 25 mM sodium citrate, pH 7.5, 2% beta mecaptoethanol, with optional detergent (5%) NP40, TX100 etc.

'''Washing Buffer''': 10 mM Tris_HCl, pH 7.5, 80% ethanol.



'''Buffers for NucleoBond AX 500 anion exchange chromatography column (Macherey-Nagel).'''

<br/>'''Resuspension buffer S1''': 50 mM Tris–HCl, 10 mM EDTA, 100 g/mL RNase A (Macherey-Nagel), pH 8.0.

<br/>'''Lysis buffer S2''': 200 mM NaOH, 1% SDS.

<br/>'''Neutralization buffer S3''': 2.8 M potassium acetate, adjusted to pH 5.1 with acetic acid.

<br/>'''Equilibration buffer N2''': 100 mM Tris–HCl, 15% ethanol, 900 mM KCl, 0.15% Triton X-100, adjusted to pH 6.3 with H3PO4.



<br/>'''Wash buffer N3''': 100 mM Tris–HCl, 15% ethanol, 1.15 M KCl, adjusted to pH 6.3 with H3PO4.

<br/>'''Elution buffer N5''': 100 mM Tris–HCl, 15% ethanol, 1 M KCl, adjusted to pH 8.5 with H3PO4.

'''N2-EF''': 100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 900 mM KCl, 0.5% Triton X-114.

'''N3-EF wash buffer:''' 100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 1150 mM KCl, 0.5% Triton X-114

ethanol: to keep Triton X-114 in solution, not turbid.

'''N4-EF''': I think it is N3-EF without Triton-114: 100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 1150 mM KCl,

'''N5-EF''' Elution Buffer: 100 mM TRIS/phosphate, pH 7.0, 15% ethanol, 1500 mM KCl.

'''S3''': 2.8 M potassium acetate, pH 5.2; stored at 4 oC.<br/>'''N2''': 900 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol.<br/>'''N3''': 1150 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol.<br/>'''N4''': 1000 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol.

=== Buffers for&nbsp;epochbiolabs.com maxiprep columns ===

VP1: 50mM Tris-HCl, pH 8.0, 10mM EDTA, With 100ug/mL RNase A; 10 ml per maxiprep

VP2: 0.2N NaOH, 1% SDS; 10 ml per maxiprep; leave at RT for 5 min to digest RNA<br/>VP3: 3M KoAc, pH 5.5

Shake column with 5 ml 98% ethanol, equilibrate column with 20 ml VPN, load supernatnat from lysate.<br/>VPN: 0.1M NaoAc, pH 5.1, 1.2M NaCl; Wash column with 30 ml VPN

VPE: 0.1M Tris-HCl, pH8.5, 1.35M NaCl, 15% Isopropanol; Elute DNA,&nbsp; 10 ml.

Precipitate DNA using 7.5 ml isopropanol, spin 30 min at 12,000 rpm. Wash pellet with 70% ethanol.

=== 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