Editing Plasmid buffers (section)

== 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]]