Plasmid buffers

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Homemade Buffers for plasmid kits


Mini-prep

[edit]



Buffer P1

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)
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 Na2EDTA.2H2O 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
Lysis Buffer: 200 mM NaOH, 1% SDS
Precipitation Buffer: 4.2 M guanidine hydrochloride, 0.9M Potassium acetate pH 4.8
Equilibration Buffer: 7M Gu-HCl pH 5.5
Wash 1 Buffer: 5M guanidine HCl, 30% Ethanol, 10 mM TRIS-HCl pH 6.6
Wash 2 Buffer: 10 mM TRIS-HCl pH 7.5, 80% Ethanol
Elution Solution: Water
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.


Buffer P2 (200mM NaOH, 1% SDS):
1M NaOH 10 ml
10% SDS 5 ml
Water 35 ml

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

Buffer P3

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

3.0 M potassium acetate pH 5.5
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% isopropanol ethanol


Buffer DP3 (for Qiagen Directprep 96-well miniprep)

3.0 M ammonium acetate pH 5.5


PB Buffer: 5 M guanidine hydrochloride, 30%isopropanol.ethanol


Buffer FWB

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


Buffer QBT

Dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) .  7g Tris-HCl, 0.5 g Tris Base in 800 ml dH2O. Adjust the pH to 7.0 with NaOH.  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
50 mM MOPS Tris-HCl, pH 7.0
15% isopropanol (v/v)
0.15% Triton® X-100 (v/v)


N3-Ca: N3 with 1.4 M CaCl2 for precipitate high molecular weight RNA

2X PEG6000-NaCl2 , 20% PEG 6000, 1.6 M NaCl.  for precipiate plasmid DNA: 4 ml of 50 % PEG 6000, 4 ml of 4 M NaCl, 2 ml H2O, autoclave.


Buffer QC

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

1.0 M NaCl
50 mM MOPS pH 7.0
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.  Add 150 ml pure isopropanol.  Adjust the volume to 1 liter with dH2O.

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


Store at 15–25°C


73.05 g NaCl

2.21g Trizma HCL

4.36 g 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.  Add 150 ml pure isopropanol.  Adjust the volume to 1 liter with dH2O.

1.6M NaCl
50 mM MOPS pH 7.0
15% isopropanol



Buffer STE

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


Buffer P3
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


4M guanidine hydrochloride (GuHCl)
0.5M potassium acetate
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

CaCl2 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


Buffer PE
80% EtOH.
25 mM NaCl,
5 mM Tris-HCl, pH 7.5

5 ml autoclaved Tris-HCl, pH 8.0

6 ml 4 M autoclaved NaCl

Autoclaved dd H2O to 250 ml

Add 800 ml EtOH

Buffer EB
10 mM Tris·Cl, pH 8-8.5
make up to 1 liter with dH20

Maxiprep

[edit]


QC buffer (wash buffer)

1.0M NaCl
50 mM MOPS pH 7.0
15% isopropanol

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

QF buffer (elution buffer)

1.25M NaCl
50 mM Tris-HCl pH 8.5
15% isopropanol

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


Buffer QN

1.6M NaCl
50 mM MOPS pH 7.0
15% isopropanol


Buffer FWB2

1M potassium acetate, pH 5.0


QBT buffer

750 mM NaCl
50 mM MOPS pH 7.0
15% isopropanol
0.15% Triton X-100

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

Buffer QBT (equilibration buffer)


P1 buffer
1M Tris HCl pH 8.0 2.5 ml
0.5 M EDTA pH 8.0 1 ml
10 mg/ml RNase A, boiled 0.5 ml
dH2O 46 ml
50ml total

P2 buffer
2M NaOH 5 ml
20% SDS 2.5 ml
dH2O 42.5 ml
50 ml total
Apparently this solution can go bad due to oxidation: keep the container tightly capped.

P3 buffer
potassium acetate 25 g
glacial acetic acid 13 ml
H2O 87 ml
100 ml total
final pH should be ~5.5


Silica membrane, low endotoxin

[edit]

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
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
50 mM EDTA pH 8.0
0.5% Tween-20
0.5% Triton-X100
RNAse A 200 ug/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 ENDO-1

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


Buffer W5-ENDO

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


Buffer W7-ENDO, Qiagen ER buffer

50 mM MOPS (pH 7.0 with NaOH)
750 mM sodium chloride
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.


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

3M NaCl
350.6 g NaCl
make up to 2 liters with dH20

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


PCR purification buffer

QXB: 5 M GHCl


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

Buffer QG 1 (neutralization and binding buffer)
5.5M guanidine thiocyanate (GuSCN)
20mM Tris-HCl
pH 6.6 (25oC)


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

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

water to 100 ml


Recipe 4
60% Guanidine thiocyanate (5.36M), 60 g
140mM MES (2-[N-Morpholino] ethanesulfonic acid)
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" alt="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%2F10.1371%2Fjournal.pone.0007750</a> 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


<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;" />
H: Homemade gel extraction buffer

Q: Qiagen gel extraction buffer QG.


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).


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


Lysis buffer S2: 200 mM NaOH, 1% SDS.


Neutralization buffer S3: 2.8 M potassium acetate, adjusted to pH 5.1 with acetic acid.


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



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


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.
N2: 900 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol.
N3: 1150 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol.
N4: 1000 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol.

Buffers for epochbiolabs.com maxiprep columns

[edit]

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
VP3: 3M KoAc, pH 5.5

Shake column with 5 ml 98% ethanol, equilibrate column with 20 ml VPN, load supernatnat from lysate.
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,  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

[edit]

Maxiprep:

Equilibration Buffer: 5~ 10%  NaOH.

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

12 ml Binding Solution (BS): 5% n-octyl-β-D-thioglucopyranoside, 8M guanidine hydrochloride
12 ml Wash Solution 1 (W1): 100 mM Tris Acetate, 4.5 M guanidine hydrochloride, 25% Isopropanol, pH 6.9
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
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

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
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 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

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


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
Fill the column with 700 μl of 1 M HCl
Cap and store in an airtight container for at least 24 hours (less than a month)
Wash the columns and collection tubes in a large beaker of water
Assemble the column and collection tube and wash the column with 700 μl of DI water, discarding the water
Repeat
Fill the column with 700 μl of buffer QBT and spin down, discarding the buffer
Place the columns in an airtight plastic bag for storage
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 » 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
==

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


Nucleobond DNA Maxi-Prep Buffers

[edit]

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

50 mM Tris pH 8.0

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


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

0.2 M NaOH
1% SDS


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

2.8 M KOAc
Glacial Acetic Acid: added until pH is 5.1


Soln Nucleobond N2: Equilibration Buffer (Store at RT)

100 mM Tris
15% EtOH
900 mM KCl
0.15% Triton X-100
Adjust with Phosphoric Acid to pH  6.3


Soln Nucleobond N3: Wash Buffer (Store at RT)

100 mM Tris
15% EtOH
1.15 M KCl
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
15% EtOH
1 M KCl
Phosphoric Acid: added until pH is 8.5



Buffer Name
 Recipe
 Buffer Name Recipe Buffer Name Recipe
 Buffer Name Recipe

  N2 1 L N3 1 L N4
 1L
 N5 1L


12.1 g 100 mM Tris 12.1 g 100 mM Tris
 12.1 g
 100 mM Tris 12.1 g

15 % EtOH 150 ml 15 % EtOH 150 ml 15 % EtOH
 150 ml
 15 % EtOH 150 ml

900 mM KCl 67.1 g 1.15 M KCl 85.7 g 1.15 M KCl
 85.73 g
 1 M KCl 74.56 g

0.15 % TX 100 1.5 ml 0.15 % TX 100 1.5 ml





pH 6.3 with H3PO4
pH 6.3 with H3PO4
pH 6.3 with H3PO4
pH 8.5 with H3PO4

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

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

N4
1150 mM KCl
100 mM Tris pH 6.3
15 Ethanol

N5
1.5M KCl
100 mM Tris pH 7.0
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.


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

N1 or L1:  29.82 g KCl
N2 or L2:  67.10 g KCl
L3: 74.55 g KCl
N3:  85.73 g KCl
N4 96.92 g KCl
N5 or L5 74.55 g KCl

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

N1 or L1 to 6.3
N2 or L2 to 6.3
N3 or L3 to 6.3
N4 to 6.3
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.)


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 12.11g of Tris base to 250 ml of deionized water and stir to dissolve.
Add 74.55g of KCl to the bring it to the required final concentration of 1.0M. Stir to dissolve and autoclave.


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.)


Adjust the pH to 8.5 with phosphoric acid.


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.)
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.
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
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
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 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
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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