Editing Plasmid buffers

Homemade Buffers for plasmid kits

== <br/><u>'''Mini-prep'''</u> ==

<br/><br/>'''Buffer P1'''<br/><br/>50 mM Tris HCl, pH 8.0, 10 mM EDTA, (or 2. 5 ml of 100X Tris-EDTA buffer from Sigma #T-9285 in 50 ml water)<br/>100 ug/ml RNase A.

'''P1''' from another protocol: 10 mM Tris-HCl, pH8, 100 ug/ml RNase A.

6.06 g Tris Base, 3.72 g Na<sub>2</sub>EDTA.2H<sub>2</sub>O in 800 ml H2O, adjust the pH with HCl, add water to 1 L, add 100 mg RNase A per liter of P1.

add some lyseblue (Thymophthalein)





'''Resuspension Buffer''': 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 ug/mL RNase A<br/>'''Lysis Buffer''': 200 mM NaOH, 1% SDS<br/>'''Precipitation Buffer''': 4.2 M guanidine hydrochloride, 0.9M Potassium acetate pH 4.8<br/>'''Equilibration Buffer''': 7M Gu-HCl pH 5.5<br/>'''Wash 1 Buffer''': 5M guanidine HCl, 30% Ethanol, 10 mM TRIS-HCl pH 6.6<br/>'''Wash 2 Buffer''': 10 mM TRIS-HCl pH 7.5, 80% Ethanol<br/>'''Elution Solution''': Water<br/>'''RNase A''': Dissolve RNASE A powder in H2O to give 100 mg/ml. Boil 5 min to kill DNase. Aliquot and store at -20 oC.<br/><br/><br/>'''Buffer P2''' (200mM NaOH, 1% SDS):<br/>1M NaOH 10 ml<br/>10% SDS 5 ml<br/>Water 35 ml

Dissolve 8.0 g NaOH in 950 ml dH2O, 50 ml 20% SDS (w/v) solution.&nbsp; The final volume should be 1 liter.<br/>Some protocols use 2% SDS.

'''Buffer P3'''

Dissolve 294.5 g potassium acetate in 500 ml dH2O.&nbsp; Adjust the pH to 5.5 with glacial acetic acid (~110 ml).&nbsp; Adjust the volume to 1 liter with dH2O.

3.0 M potassium acetate pH 5.5<br/>can be stored either at room temp or 2~8°C



'''PB'''

5 M guanidine hydrochloride (MW 95.53 ), 20 mM Tris-HCl, pH 6.6, (25 oC), add ethanol to 38%.

200 ml:

95.53 g guanidine hydrochloride

8 ml of 0.5M Tris-HCl, pH6.6

76 ml of ethanol



'''PB from another protocol:''' 5 M Gu-HCl, 30% <strike>isopropanol</strike> ethanol

<br/>'''Buffer DP3''' (for Qiagen Directprep 96-well miniprep)

3.0 M ammonium acetate pH 5.5



'''PB Buffer''': 5 M guanidine hydrochloride, 30%<strike>isopropanol</strike>.ethanol



'''Buffer FWB'''

Dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml dH2O.&nbsp; Adjust the pH to 7.0 with NaOH.&nbsp; Add 150 ml pure isopropanol and adjust the volume to 1 liter with dH2O.



'''Buffer QBT'''

Dissolve 43.83 g NaCl, <strike>10.46 g MOPS (free acid) .</strike>&nbsp; 7g Tris-HCl, 0.5 g Tris Base in 800 ml dH2O. <strike>Adjust the pH to 7.0 with NaOH.&nbsp;</strike> Add 150 ml pure isopropanol and 15 ml 10% Triton X-100 solution (v/v). Adjust the volume to 1 liter with dH2O.

750 mM NaCl<br/>50 mM <strike>MOPS </strike>Tris-HCl, pH 7.0<br/>15% isopropanol (v/v)<br/>0.15% Triton® X-100 (v/v)



'''N3-Ca''': N3 with 1.4 M CaCl<sub>2</sub>&nbsp;for precipitate high molecular weight&nbsp;RNA

'''2X PEG6000-NaCl<sub>2</sub>'''&nbsp;, 20% PEG 6000, 1.6 M NaCl.&nbsp;&nbsp;for precipiate plasmid DNA: 4 ml of 50&nbsp;% PEG 6000, 4 ml of 4 M NaCl, 2 ml H<sub>2</sub>O, autoclave.



'''Buffer QC'''

Dissolve 58.44 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O.&nbsp; Adjust the pH to 7.0 with NaOH.&nbsp; Add 150 ml pure isopropanol.&nbsp; Adjust the volume to 1 liter with dH2O.

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



'''Buffer QF'''

'''From Qiagen website'''

Dissolve 73.05 g NaCl and 6.06 g Tris base in 800 ml dH2O and adjust the pH to 8.5 with HCl.&nbsp; Add 150 ml pure isopropanol.&nbsp; Adjust the volume to 1 liter with dH2O.

1.25 M NaCl<br/>50 mM Tris·Cl pH 8.5<br/>15% isopropanol (v/v)



Store at 15–25°C


73.05 g&nbsp;NaCl

2.21<span style="white-space:pre">g </span>Trizma HCL

4.36 g&nbsp;Trizma Base



'''Buffer QN'''

'''from Qiagen website'''

Dissolve 93.50 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O and adjust the pH to 7.0 with NaOH.&nbsp; Add 150 ml pure isopropanol.&nbsp; Adjust the volume to 1 liter with dH2O.

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





'''Buffer STE'''

Dissolve 5.84 g NaCl, 1.21 g Tris base, and 0.37 g Na2EDTA*2H2O in 800 ml dH2O.&nbsp; Adjust the pH to 8.0 with HCl.&nbsp; Adjust the volume to 1 liter with dH2O.

<br/>'''Buffer P3'''<br/>3.0 M KOAc, pH 5.5 (29.5 g/ 100ml water, adjust pH with glacial acetic acid )





'''N3 from another protocol''':

4.2 M guanidine hydrochloride, 0.9M KOAc, pH 4.8, adjust to pH4.2~4.8 by adding acetic acid.

200 ml:

80 g guanidine hydrochloride

18g potassium acetate

10 ml acetic acid (add this inside the hood),

add DI water to dissolve

add water to 200 ml.



Yet another protocol

<br/>4M guanidine hydrochloride (GuHCl)<br/>0.5M potassium acetate<br/>pH 4.2



'''Improved N3''' 4.2 M guanidine hydrochloride, 0.9M KOAc, pH 4.8, adjust to pH4.2~4.8 by adding acetic acid.

200 ml:

80 g guanidine hydrochloride

18g potassium acetate

10 ml acetic acid (add this inside the hood),

'''6 ml of 1 M MgCl2'''

'''CaCl<sub>2</sub> 1.4 M'''



add DI water to dissolve

add water to 200 ml.

Buffer N3

4.2 M Gu-HCl 0.9 M potassium acetate pH 4.8 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

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

5 M GuHCl

<br/>'''Buffer PE'''<br/>80% EtOH.<br/>25 mM NaCl,<br/>5 mM Tris-HCl, pH 7.5

5 ml autoclaved Tris-HCl, pH 8.0

6 ml 4 M autoclaved NaCl

Autoclaved dd H<sub>2</sub>O to 250 ml

Add 800 ml EtOH<br/><br/>'''Buffer EB'''<br/>10 mM Tris·Cl, pH 8-8.5<br/>make up to 1 liter with dH20

== <u>'''Maxiprep'''</u> ==

<br/>'''QC buffer''' (wash buffer)

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

to make 2 L<br/>666 ml 3M NaCl<br/>200 ml 0.5 M MOPS pH 7.0<br/>300 ml EtOH<br/>dH2O to 2 liters<br/>check the pH and adjust to 7.0 (it should be close) with NaOH or HCl

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

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

to make 2 L<br/>833 ml 3M NaCl<br/>100 ml 1M Tris pH 8.5<br/>300 ml EtOH<br/>dH2O to 2 liters<br/>check the pH and adjust to 8.5 (it should be close) with NaOH or HCl<br/>store QC and QF in tightly capped containers at room temp.



'''Buffer QN'''

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



'''Buffer FWB2'''

1M potassium acetate, pH 5.0



'''QBT buffer'''

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

to make 2 L:<br/>500 ml 3M NaCl<br/>200 ml 0.5 M MOPS pH 7.0<br/>300 ml EtOH<br/>3 ml Triton X-100<br/>dH2O to 2 liters

Buffer QBT (equilibration buffer)



'''P1 buffer'''<br/>1M Tris HCl pH 8.0 2.5 ml<br/>0.5 M EDTA pH 8.0 1 ml<br/>10 mg/ml RNase A, boiled 0.5 ml<br/>dH2O 46 ml<br/>50ml total<br/><br/>'''P2 buffer'''<br/>2M NaOH 5 ml<br/>20% SDS 2.5 ml<br/>dH2O 42.5 ml<br/>50 ml total<br/>Apparently this solution can go bad due to oxidation: keep the container tightly capped.<br/><br/>'''P3 buffer'''<br/>potassium acetate 25 g<br/>glacial acetic acid 13 ml<br/>H2O 87 ml<br/>100 ml total<br/>final pH should be ~5.5



== 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



== Nucleobond DNA Maxi-Prep Buffers ==

Soln P1 or S1: TE Resuspension Buffer (Store at 4 °C)

50 mM Tris pH 8.0

10 mM EDTA (8.0)<br/>0.1 mg/mL (100ug/ml) RNAse A



'''Soln P2 or S2''': NaOH/SDS Lysis Buffer (Store at RT)

0.2 M NaOH<br/>1% SDS



'''Soln P3 or S3''': KOAC Neutralization Buffer (Store at 4 °C)

2.8 M KOAc<br/>Glacial Acetic Acid: added until pH is 5.1



Soln Nucleobond '''N2''': Equilibration Buffer (Store at RT)

100 mM Tris<br/>15% EtOH<br/>900 mM KCl<br/>0.15% Triton X-100<br/>Adjust with&nbsp;Phosphoric Acid to pH&nbsp; 6.3



Soln Nucleobond '''N3''': Wash Buffer (Store at RT)

100 mM Tris<br/>15% EtOH<br/>1.15 M KCl<br/>Phosphoric Acid: added until pH is 6.3



'''ENDO-EF''':

It has ethanol and Triton X-100 or Triton X-114.

100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 1150 mM KCl, 0.5% Triton X-114



'''N5-EF'''

100 mM TRIS/phosphate, pH 6.8~7.0, or 0.1~0.2 M Phosphate Buffer, pH 6.8~7.0, 1.8 to 2 M NaCl (or 1.8 M KCl).





Soln Nucleobond '''N5''': Elution Buffer (Store at RT)

100 mM Tris<br/>15% EtOH<br/>1 M KCl<br/>Phosphoric Acid: added until pH is 8.5



{|
|-
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | Buffer Name<br/>
| bgcolor="#ffffcc" | Recipe<br/>
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe<br/>
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe
|-
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | &nbsp; N2
| bgcolor="#ffffcc" | 1 L
| bgcolor="#ffffcc" | N3
| bgcolor="#ffffcc" | 1 L
| bgcolor="#ffffcc" | N4<br/>
| bgcolor="#ffffcc" | 1L<br/>
| bgcolor="#ffffcc" | N5
| bgcolor="#ffffcc" | 1L
|-
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | 12.1 g
| bgcolor="#ffffcc" | 100 mM Tris
| bgcolor="#ffffcc" | 12.1 g
| bgcolor="#ffffcc" | 100 mM Tris<br/>
| bgcolor="#ffffcc" | 12.1 g<br/>
| bgcolor="#ffffcc" | 100 mM Tris
| bgcolor="#ffffcc" | 12.1 g
|-
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | 15&nbsp;% EtOH
| bgcolor="#ffffcc" | 150 ml
| bgcolor="#ffffcc" | 15&nbsp;% EtOH
| bgcolor="#ffffcc" | 150 ml
| bgcolor="#ffffcc" | 15&nbsp;% EtOH<br/>
| bgcolor="#ffffcc" | 150 ml<br/>
| bgcolor="#ffffcc" | 15&nbsp;% EtOH
| bgcolor="#ffffcc" | 150 ml
|-
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | 900 mM KCl
| bgcolor="#ffffcc" | 67.1 g
| bgcolor="#ffffcc" | 1.15 M KCl
| bgcolor="#ffffcc" | 85.7 g
| bgcolor="#ffffcc" | 1.15 M KCl<br/>
| bgcolor="#ffffcc" | 85.73 g<br/>
| bgcolor="#ffffcc" | 1 M KCl
| bgcolor="#ffffcc" | 74.56 g
|-
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | 0.15&nbsp;% TX 100
| bgcolor="#ffffcc" | 1.5 ml
| bgcolor="#ffffcc" | 0.15&nbsp;% TX 100
| bgcolor="#ffffcc" | 1.5 ml
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | <br/>
|-
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4<br/>
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4<br/>
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4<br/>
| bgcolor="#ffffcc" | <br/>
| bgcolor="#ffffcc" | pH '''8.5''' with H3PO4<br/>
|}

N2<br/>900 mMKCl<br/>100 mM Tri pH6.3<br/>15% Ethanol<br/>0.5% Triton X-114

N3<br/>1150 mM KCl<br/>100 mM Tris pH 6.3<br/>15% Ethanol<br/>0.5% Triton X-114

N4<br/>1150 mM KCl<br/>100 mM Tris pH 6.3<br/>15 Ethanol

N5<br/>1.5M KCl<br/>100 mM Tris pH 7.0<br/>15% Ethanol

'''N and L Series Buffer Preparation for Normal Processing of Nucleobond AX Cartridges'''

The most important factors when making up the N or L series of buffers for Nucleobond AX cartridges are the concentration of KCl and the pH. Even slight deviations in these could change the elution profiles of different nucleic acids. The following guidelines are presented primarily for those laboratories who use Nucleobond AX cartridges with buffers that they choose to make themselves. The protocol assumes that the buffers and cartridges will be used for both DNA and RNA purifications. If only DNA is to be purified, the DEPC treatment in step 1 can be omitted. The following procedure will make one liter of each buffer.

Prepare DEPC (diethylpyrocarbonate)-treated water by adding 0.1 ml DEPC per 100 ml distilled water. Stir for at least two hours and autoclave for 30-40 minutes to obtain RNAse-free sterile water.

<br/>For each N or L solution, begin by adding 12.11 g of Tris base to 600 ml of water and stir to dissolve.<br/>Add KCl to the required final concentration for each buffer as follows:

N1 or L1:&nbsp; 29.82 g KCl<br/>N2 or L2:&nbsp; 67.10 g KCl<br/>L3: 74.55 g KCl<br/>N3:&nbsp; 85.73 g KCl<br/>N4 96.92 g KCl<br/>N5 or L5 74.55 g KCl

Stir to dissolve and autoclave.<br/>Allow the autoclaved buffers to cool to room temperature and add 150 ml of absolute (100%) ethanol to each. (See NOTE)<br/>Adjust the pH of each buffer with phosphoric acid as follows:

N1 or L1 to 6.3<br/>N2 or L2 to 6.3<br/>N3 or L3 to 6.3<br/>N4 to 6.3<br/>N5 or L5 to 8.5

(Be very careful not to "overshoot" the appropriate pH for each solution when titrating because correcting it with a basic solution will alter the salt concentration of the buffer which, in turn, could alter cartridge performance.)

<br/>For each buffer add enough autoclaved, distilled water (DEPC-treated, if required) to reach a final volume of 1000 ml. Store at room temperature in a tightly closed container to prevent evaporation.



NOTE 1: The ethanol and phosphoric acids available to most laboratories are DNAse/RNAse free. This can be easily checked for those used in your lab. Dilute the ethanol or acid approximately 10 to 1 in DEPC-treated water and add MgCl2 to a final concentration of 10 mM. Add 1-2 mg of RNA (or DNA), incubate at 37C for 30 minutes, and run on a 1.2% agarose gel (prepared with DEPC-treated water and RNAse-free loading and running buffers, if necessary) to determine whether the RNA (or DNA) was degraded. A control lane of RNA (or DNA) in DEPC-treated water alone will determine if any degradation resulted from the gel itself.





'''N5 and L5 Elution Buffer Preparation for High GC Content DNA'''

DNA with GC contents higher than 60% (or with large regions of high GC content) may continue to bind a Nucleobond AX cartridge even under normal elution conditions. In such cases, some type of DNA denaturant must be added in order to do the job. The final buffer composition that has been shown to work for the modified N5 and L5 elution buffers is as follows: 50% formamide, 1.0 M KCl, 15% EtOH, 100 mM Tris-phosphate, pH 8.5. The following procedure will make one liter of this buffer.

Begin by adding <u>12.11g</u> of Tris base to 250 ml of deionized water and stir to dissolve.<br/>Add <u>74.55g of KC</u>l to the bring it to the required final concentration of 1.0M. Stir to dissolve and autoclave.

<br/>Allow the autoclaved buffer to cool to room temperature and add 150 ml of absolute (100%) ethanol1 and 500 ml of deionized formamide. (The formamide can be deionized by treating it with a mixed bed resin to bring the pH close to 7.)

<br/>Adjust the pH to 8.5 with phosphoric acid.

<br/>NOTE: The ethanol, formamide, and phosphoric acids available to most laboratories are DNAse/RNAse free. This can be easily checked for those used in your lab. Dilute the reagent approximately 10 to 1 in DEPC-treated water and add MgCl2 to a final concentration of 10 mM. Add 1-2 mg of RNA (or DNA), incubate at 37C for 30 minutes, and run on a 1.2% agarose gel (prepared with DEPC-treated water and RNAse-free loading and running buffers, if necessary) to determine whether the RNA (or DNA) was degraded. A control lane of RNA (or DNA) in DEPC-treated water alone will determine if any degradation resulted from the gel itself.

(Be very careful not to "overshoot" the appropriate pH for each solution when titrating because correcting it with a basic solution will alter the salt concentration of the buffer which, in turn, could alter cartridge performance.)<br/>Add enough autoclaved, distilled water to reach a final volume of 1000 ml. Store at room temperature in a tightly closed container to prevent evaporation.



Formulas for Qiagen Kit Buffers

Do not autoclave solutions containing ethanol, isopropanol or MOPS; use sterile filtration if necessary.<br/>Buffer AE (elution buffer for genomic DNA preps)<br/>10 mM Tris-HCl<br/>0.5 mM EDTA<br/>pH 9.0<br/>Buffer P1<br/>50 mM Tris-HCl pH 8.0<br/>10 mM EDTA<br/>100 μg/ml RNaseA<br/>The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101).<br/>Buffer P2<br/>200 mM NaOH<br/>1% SDS<br/>Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits)<br/>3.0 M potassium acetate pH 5.5<br/>Buffer DP3 (for Qiagen Directprep 96-well miniprep)<br/>3.0 M ammonium acetate pH 5.5<br/>Buffer N3<br/>4.2 M Gu-HCl<br/>0.9 M potassium acetate<br/>pH 4.8<br/>Buffer PB<br/>5 M Gu-HCl<br/>30% isopropanol<br/>Buffer QG<br/>5.5 M guanidine thiocyanate (GuSCN)<br/>20 mM Tris HCl pH 6.6<br/>Buffer PE<br/>10 mM Tris-HCl pH 7.5<br/>80% ethanol<br/>Buffer QX1 (for solution and binding of agarose gels)<br/>7 M NaPO4<br/>10 mM NaAc<br/>pH 5.3<br/>Buffer QXB (for binding of large >3000 bp fragments to columns)<br/>5 M GuHCl<br/>Buffer QBT (equilibration buffer)<br/>750 mM NaCl<br/>50 mM MOPS pH 7.0<br/>15% isopropanol<br/>0.15% triton X-100<br/>Buffer QC (wash buffer)<br/>1.0M NaCl<br/>50 mM MOPS pH 7.0<br/>15% isopropanol<br/>Buffer QF (elution buffer)<br/>1.25M NaCl<br/>50 mM Tris-HCl pH 8.5<br/>15% isopropanol<br/>Buffer QN<br/>1.6M NaCl<br/>50 mM MOPS pH 7.0<br/>15% isopropanol<br/>Buffer FWB2<br/>1M potassium acetate, pH 5.0<br/>Buffer B1 (bacterial lysis buffer)<br/>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<br/>Buffer B2 (bacterial lysis buffer)<br/>3 M Gu-HCl<br/>20% Tween-20<br/>Buffer C1 (cell lysis buffer) (store at +4)<br/>1.28 M sucrose<br/>40 mM Tris-HCl pH 7.5<br/>20 mM MgCl2<br/>4% Triton X-100<br/>Buffer G2 (digestion buffer)<br/>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<br/>Buffer Y1 (yeast lysis buffer) (store at +4)<br/>1 M Sorbitol<br/>100 mM EDTA pH 8.0<br/>14 mM beta mercaptoethanol (added just before use)<br/>Buffer PAA (PAGE gel elution of DNA)<br/>500 mM NH4Ac<br/>100 mM MgAc2<br/>1 mM EDTA<br/>0.1% SDS<br/>Buffer PNI (purification of oligonucleotides 17 nt and greater)<br/>40% (v/v) 5 M guanidinium chloride<br/>60% (v/v) isopropanol<br/>&nbsp;Concentration of guanidinium determined by drying down supplied concentrate and measuring mass. Concentration of isopropanol determined by calculation from the supplied user manual.&nbsp;

(Source: [1], US Patent 6,383,393)<br/>The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3
[[Category:Buffer Recipe]]