https://wiki.phagocytes.ca/index.php?action=history&feed=atom&title=Common_buffers
Common buffers - Revision history
2026-04-16T08:03:54Z
Revision history for this page on the wiki
MediaWiki 1.40.1
https://wiki.phagocytes.ca/index.php?title=Common_buffers&diff=13&oldid=prev
Admin: Created page with "= Cell Culture = ===== 1x PBS<br> ===== To 800ml ddH<sub>2</sub>O add:<br> *8g NaCl<br> *0.2g KCl<br> *1.44g Na<sub>2</sub>HPO<sub>4</sub> (sodium phosphate dibasic)<br>..."
2021-02-01T19:25:01Z
<p>Created page with "= Cell Culture = ===== 1x PBS<br> ===== To 800ml ddH<sub>2</sub>O add:<br> *8g NaCl<br> *0.2g KCl<br> *1.44g Na<sub>2</sub>HPO<sub>4</sub> (sodium phosphate dibasic)<br>..."</p>
<p><b>New page</b></p><div>= Cell Culture =<br />
<br />
===== 1x PBS<br> =====<br />
<br />
To 800ml ddH<sub>2</sub>O add:<br><br />
<br />
*8g NaCl<br> <br />
*0.2g KCl<br> <br />
*1.44g Na<sub>2</sub>HPO<sub>4</sub> (sodium phosphate dibasic)<br> <br />
*0.24g KH<sub>2</sub>PO<sub>4</sub> (monopotassium phosphate)<br> <br />
<br />
pH to 7.4, and bring up to 1L with ddH<sub>2</sub>O. Autoclave to sterilize.<br><br />
<br />
===== 10x PBS<br> =====<br />
<br />
To 800ml ddH<sub>2</sub>O add:<br><br />
<br />
*80g NaCl<br> <br />
*2.0g KCl<br> <br />
*14.4g Na<sub>2</sub>HPO<sub>4</sub> (sodium phosphate dibasic)<br> <br />
*2.4g KH<sub>2</sub>PO<sub>4</sub> (monopotassium phosphate)<br> <br />
<br />
pH to 7.4, and bring up to 1L with ddH<sub>2</sub>O. Autoclave to sterilize.<br><br />
<br />
===== 50x TAE<br> =====<br />
<br />
For each litre of solution:<br />
<br />
*242g Tris Base<br />
*57.1ml Glacial Acetic Acid<br />
*100ml 0.5M EDTA<br />
<br />
#Mix Tris with stir bar to dissolve in about 600ml of ddH<sub>2</sub>O.<br />
#Add the EDTA and Acetic Acid.<br />
#Bring final volume to 1L with ddH<sub>2</sub>O.<br />
#Store at room temperature.<br />
<br />
'''Note:''' Final (1x) working concentration:<br />
<br />
*0.04M Tris-Acetate<br />
*0.001M EDTA<br />
<br />
= Western Blotting =<br />
<br />
===== Lysis Buffer =====<br />
<br />
{| cellspacing="1" cellpadding="1" border="1"<br />
|- valign="center"<br />
| '''Reagent'''<br><br />
| '''Stock Solution<br>'''<br />
| '''Volume'''<br><br />
|-<br />
| ddH<sub>2</sub>O<br><br />
| <center>--</center><br />
| <center>7.68ml</center><br />
|- valign="center"<br />
| Tris, pH 8.0<br><br />
| <center>20mM</center> <br />
| <center>200ul</center> <br />
|- valign="center"<br />
| NaCl<br><br />
| <center>0.15M</center> <br />
| <center>750ul</center> <br />
|- valign="center"<br />
| EDTA<br><br />
| <center>2mM</center> <br />
| <center>40ul</center> <br />
|- valign="center"<br />
| NP40<br><br />
| <center>--</center> <br />
| <center>100ul</center> <br />
|- valign="center"<br />
| Glycerol<br><br />
| <center>--</center> <br />
| <center>1000ul</center> <br />
|- valign="center"<br />
| Na<sub>3</sub>VO<sub>4</sub><br><br />
| <center>1mM</center> <br />
| <center>100ul</center> <br />
|}<br />
<br />
The above recipe is for 10ml.&nbsp; This buffer can be made in bulk and used as a wash buffer, or allotted and frozen for future use.&nbsp; Immediately before use add protease inhibitors at the manufacturers recommended concentration, and phosphatase inhibitors if required.<br><br />
<br />
===== Laemmli's Buffer, 4x =====<br />
<br />
*2.4 ml 1 M Tris pH 6.8 (Same as upper gel buffer) <br />
*0.8 g SDS stock <br />
*4 ml 100% glycerol <br />
*0.01% bromophenol blue. Final Concentration is .02% <br />
*2.8 ml ddH<sub>2</sub>O <br />
<br />
Before use add 1/10<sup>th</sup> volume of β-mercaptoethanol<br />
<br />
===== Laemmli's Buffer, 6x =====<br />
<br />
*1.2g SDS (sodium dodecyl sulfate) <br />
*0.01% bromophenol blue <br />
*4.7ml glycerol <br />
*1.2ml Tris 0.5M pH6.8 <br />
*2.1ml ddH<sub>2</sub>O<br> <br />
<br />
Before use add 1/8<sup>th</sup> volume of β-mercaptoethanol<br />
<br />
===== 4x Lower Gel Buffer =====<br />
<br />
*182g Tris-Base (1.5M) <br />
*1L dH2O<br> <br />
<br />
pH to 8.8; do not overshoot. Use glass pipettes to pH.<br />
<br />
===== 4x Upper Gel Buffer =====<br />
<br />
*30.28g Tris-HCl (0.5M) <br />
*500ml dH2O <br />
<br />
pH to 6.8, do not overshoot. Use glass pipettes.<br />
<br />
===== 10x Running Buffer<br> =====<br />
<br />
*30.4g Tri-HCl <br />
*144.2g Glycine <br />
*10g SDS <br />
<br />
Dissolve in 1L dH2O. Dilute 1:10 in ddH2O for use as running buffer<br />
<br />
===== 10X Transfer Buffer (Towbin Buffer) =====<br />
<br />
*30.3g Tris-Base <br />
*144.15g Glycine <br />
*100ml 10% SDS (0.1%) <br />
<br />
Bring upto 1L in ddH2O.<br><br />
<br />
===== 1X Transfer Buffer (Towbin Buffer) =====<br />
<br />
*100ml 10X buffer <br />
*200ml methanol <br />
*700ml ddH<sub>2</sub>O <br />
<br />
===== 10x TBS =====<br />
<br />
*302.5g Tris-Base <br />
*400g NaCl <br />
*18g KCl <br />
<br />
Dilute to 5L in dH2O, pH to 7.5 with saturated HCl.<br><br />
<br />
===== Wash Buffer (TBST) =====<br />
<br />
to 1X TBS ADD:<br />
<br />
{| width="200" cellspacing="1" cellpadding="1" border="1"<br />
|-<br />
| Standard<br><br />
| 0.1% Tween-20<br><br />
|-<br />
| Strong<br><br />
| 0.1% Tween-20 + 0.1% NP-40<br><br />
|-<br />
| Extra-Strong<br><br />
| 0.1% Tween-20 + 1.5% NP-40<br><br />
|}<br />
<pre>Note: Standard buffer is for most application. Strong/extra strong are only for antibodies with a high degree of non-specificity.<br />
</pre><br />
<br />
= DNA Buffers =<br />
===== 6X DNA Loading Buffer =====<br />
<br />
*6.7 ml of ddH2O <br />
*10 mg of bromophenol blue <br />
*10 mg of xylene cyanol FF (optional) <br />
*3.3ml glycerol <br />
<br />
''Notes:''<br />
<br />
#Bromophenol Blue runs at ~300bp (should be added to avoid over-running of gels) <br />
#Xylene cyanol FF runs at ~4000bp (optional dye) <br />
#60 mM EDTA can be added to inhibit DNA-altering enzymes (600ul of 1M EDTA; reduce water appropriately)</div>
Admin