{"id":90,"date":"2020-05-21T22:04:39","date_gmt":"2020-05-21T21:04:39","guid":{"rendered":"https:\/\/www.adras.xyz\/aswin\/?page_id=90"},"modified":"2022-05-18T14:10:05","modified_gmt":"2022-05-18T13:10:05","slug":"useful-buffers","status":"publish","type":"page","link":"https:\/\/www.adras.xyz\/aswin\/tools\/useful-buffers\/","title":{"rendered":"Useful buffers"},"content":{"rendered":"\n

Biological buffers<\/b>

Tris buffer<\/i>
The established protocol for ensilication requires 50 mM of Tris buffer at pH 7.0 with no sodium present. Therefore, 3.03 g of Trizma-Base (MW: 121.14 g\/mol, cat: T666-1kg, Sigma UK) was weighed out to make 500 ml buffer. In a beaker, 400 ml of ultrapure (MilliQ, MilliPore UK) water was added to the weighed Trizma and using a magnetic stirrer this was dissolved. The pH was set using 32% (v\/v) hydrochloric acid. Finally, the solution was filled up to 500 ml final volume with ultrapure water.

Recombinant expression in E. coli<\/i>
Bacterial medium, Luria Broth<\/i> (LB) contains 10 g\/L Tryptone, 5 g\/L Yeast Extract, 5 g\/L Sodium Chloride, Sigma, in ddH2<\/sub>O. Autoclave after preparation and use when needed.

Agar plates were made of LB, 10 g\/L Tryptone, 5 g\/L Yeast Extract, 10 g\/L Sodium Chloride, with 15 g\/L added agarose, Sigma, in ddH2<\/sub>O. Autoclave after preparation and pour plates when hand warm in a laminar flow-hood.

Bacterial stocks were made by addition of 30% glycerol and stored at -80 \u00b0C until further use.

Buffers for HisTag purification: <\/em><\/p>\n\n\n\n

<\/td> NaCl <\/b><\/td> Tris<\/b><\/td> Imidazole<\/b><\/td><\/td><\/tr>
IMAC25<\/sub> (binding)<\/td> 0.5 M <\/td> 0.1 M<\/td> 25 mM <\/td>pH 8.0 in ddH2<\/sub>O.<\/td><\/td><\/tr>
IMAC500<\/sub> (elution)<\/td> 0.5 M <\/td> 0.1 M<\/td> 500 mM <\/td>pH 8.0 in ddH2<\/sub>O.<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

pH solution with HCl. Autoclave or filter 0.45 \u03bcm for use in the FPLC.

ELISA 5\u2019-5\u2019 tetra-methyl-benzidine (TMB, Sigma, UK) substrate <\/i><\/p>\n\n\n\n

<\/td>TMB<\/b><\/td>NaAct<\/b><\/td>H2<\/sub>O2<\/sub><\/b><\/td><\/td><\/tr>
stock<\/td>10 \u03bcg \/ ml<\/td>0.1 M pH 6.0<\/td>30 % (v\/v)<\/td> TMB in DMSO or EtOH
NaAct stock 1M pH with citric acid.<\/td><\/tr>
in 10 ml<\/td>100 \u00b5L<\/td>10 ml<\/td>1.5 \u00b5L<\/td><\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

\n\n5\u2019-5\u2019 tetra-methyl-benzidine (TMB, Sigma, UK) was dissolved in 1 ml of dimethylsulfoxide (DMSO, Sigma, UK) or EtOH at 10 \u03bcg \/ ml and 100 \u03bcL was added to 10 ml of 0.1M sodium acetate pH 6.0 (with citric acid) with addition of 1.5 \u03bcL of 30% (v\/v) H2<\/sub>O2<\/sub>.\n<\/p>\n\n\n\n

Sodium Phosphate Buffers<\/em><\/p>\n\n\n\n

pH<\/th>Volume (mL) of 1 M Na2<\/sub>HPO4<\/sub><\/th>Volume (mL) of 1 M NaH2<\/sub>PO4<\/sub><\/th><\/tr><\/thead>
5.8<\/td>7.9<\/td>92.1<\/td><\/tr>
6.0<\/td>12.0<\/td>88.0<\/td><\/tr>
6.2<\/td>17.8<\/td>82.2<\/td><\/tr>
6.4<\/td>25.5<\/td>74.5<\/td><\/tr>
6.6<\/td>35.2<\/td>64.8<\/td><\/tr>
6.8<\/td>46.3<\/td>53.7<\/td><\/tr>
7.0<\/td>57.7<\/td>42.3<\/td><\/tr>
7.2<\/td>68.4<\/td>31.6<\/td><\/tr>
7.4<\/td>77.4<\/td>22.6<\/td><\/tr>
7.6<\/td>84.5<\/td>15.5<\/td><\/tr>
7.8<\/td>89.6<\/td>10.4<\/td><\/tr>
8.0<\/td>93.2<\/td>6.8<\/td><\/tr><\/tbody><\/table><\/figure>\n","protected":false},"excerpt":{"rendered":"

Biological buffers Tris buffer The established protocol for ensilication requires 50 mM of Tris buffer at pH 7.0 with no sodium present. Therefore, 3.03 g of Trizma-Base (MW: 121.14 g\/mol, cat: T666-1kg, Sigma UK) was weighed out to make 500 ml buffer. In a beaker, 400 ml of ultrapure (MilliQ, MilliPore UK) water was added […]<\/p>\n","protected":false},"author":1,"featured_media":0,"parent":29,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-templates\/child-menu.php","meta":[],"_links":{"self":[{"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/pages\/90"}],"collection":[{"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/comments?post=90"}],"version-history":[{"count":9,"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/pages\/90\/revisions"}],"predecessor-version":[{"id":212,"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/pages\/90\/revisions\/212"}],"up":[{"embeddable":true,"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/pages\/29"}],"wp:attachment":[{"href":"https:\/\/www.adras.xyz\/aswin\/wp-json\/wp\/v2\/media?parent=90"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}