User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/01: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
(fix raw html notebook nav) |
||
(7 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==Tasks for October 1== | ==Tasks for October 1== | ||
* Analysis of Dialysis Solutions | * Analysis of Dialysis Solutions from [http://openwetware.org/wiki/User:Alicia_Rasines_Mazo/Notebook/CHEM-571_Experimental_Biological_Chemistry/2014/09/30 Sept. 30] | ||
===Analysis of Dialysis Solution=== | ===Analysis of Dialysis Solution=== | ||
Procedure followed as detailed by [http://openwetware.org/wiki/User:Douglas_M._Fox/Notebook/AU_CHEM-571_F2011_Lab_Support/2014/10/01 Dr. Fox on Oct.1] | Procedure followed as detailed by [http://openwetware.org/wiki/User:Douglas_M._Fox/Notebook/AU_CHEM-571_F2011_Lab_Support/2014/10/01 Dr. Fox on Oct.1] | ||
* Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis | |||
**Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl | |||
**Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer | |||
**PS cuvettes, measuring 400 - 800 nm | |||
**Don't forget to run a blank with just Bradford & buffer | |||
**Don't forget to run your undialyzed Lysozyme stock | |||
* Transfer remainder of each cell into 20 mL extraction vials | |||
* Measure Ca<sup>2+</sup> using ISE | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Substance''' | |||
| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)''' | |||
|- | |||
| 50mM CaCl<sub>2</sub> (4) || 80.8 | |||
|- | |||
| Lysozyme (4) || 80.3 | |||
|- | |||
| 500μM CaCl<sub>2</sub> (3)||28.8 | |||
|- | |||
| Lysozyme (3) || 28.1 | |||
|- | |||
|} | |||
* Transfer 100 μL to a small volume UV cuvette & measure UV absorption | |||
**Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes | |||
**Measure entire 200 - 800 nm range | |||
* Transfer 100 μL to a small volume fluorescence cuvette & measure fluorescence | |||
**Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes | |||
**excitation at 280 nm | |||
* Transfer 500 - 700 μL to a 15 mL Falcon tube. Add 2 - 3 times volume of water & measure pH | |||
**You want to add minimum amount of water | |||
**You typically need 1.6 - 2 mL in falcon tube to cover pH probe | |||
**So, if you have only 500 μL left, you'll need a 4x dilution (adding 1.5 mL water) | |||
**Or, if you have 700 μL left, you only need a 3x dilution (adding 1.4 mL water) | |||
===Preparation of new dialysis=== | |||
A new dialysis experiment was prepared using '''22,000 MWCO tubing''' instead of 3500MWCO. <br.> | |||
The wells were matched up in the following way. Note that 1 mL of the following were added to each well: | |||
# 50 mM CaCl<sub>2</sub>. Opposite to it Colloid 1 | |||
# 500 μM CaCl<sub>2</sub>. Opposite to it Colloid 2 | |||
# HPLC water. Opposite to it Lysozyme 3 | |||
# 0.25 mM HCl. Opposite to it Lysozyme 4 | |||
# 50 mM NaCl. Opposite to it Lysozyme 5 | |||
* Inserted screws to prevent evaporation | |||
* Placed on low speed shaker for 1 week |
Latest revision as of 00:22, 27 September 2017
Project name | Main project page Previous entry Next entry | ||||||||||
Tasks for October 1
Analysis of Dialysis SolutionProcedure followed as detailed by Dr. Fox on Oct.1
Preparation of new dialysisA new dialysis experiment was prepared using 22,000 MWCO tubing instead of 3500MWCO. <br.> The wells were matched up in the following way. Note that 1 mL of the following were added to each well:
|