User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2013/02/27

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(Autocreate 2013/02/27 Entry for User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I)
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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Experimental Biological Chemistry II</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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| 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. ##### -->
-
==Entry title==
+
==Purpose==
-
* Insert content here...
+
* Run pilot study on cell growth with concentrations of AgNO<sub>3</sub> in solution varying from 1uM to 10mM
 +
 
 +
==Procedure==
 +
* DH5α-T1 cell pellets were taken out from -80°C freezer and left in room temperature to defrost.
 +
* Under a sterile environment, 3mL of autoclaved LB medium were added onto pellet. The solution was pipetted up and down gently to mix.
 +
* In five bottles of 125mL Erlenmeyer flasks containing 50mL LB medium, the following amount of LB were taken out of the medium from each flask:
 +
  4.76uL, 5.00uL, 50.0uL, 500uL, 5000uL
 +
* Respectively, the following amount and concentrations of AgNO<sub>3</sub> were placed in:
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Volume of LB taken out(uL)'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume of AgNO3 added(uL)'''
 +
| align="center" style="background:#f0f0f0;"|'''Concentration of AgNO3 added(M)'''
 +
| align="center" style="background:#f0f0f0;"|'''Final Concentration of AgNO3 in flask(uM)'''
 +
|-
 +
| 4.76||4.76||0.0105||1
 +
|-
 +
| 5||5||0.1||10
 +
|-
 +
| 50||50||0.1||100
 +
|-
 +
| 500||500||0.1||1,000
 +
|-
 +
| 5000||5000||0.1||10,000
 +
|}
 +
* Then, 1mL of resuspended cells were placed into each of the five flasks.
 +
* The positive control contains 50mL of LB media and 1mL of cells
 +
* One more control was set up containing 45mL of LB media and 5mL of 0.1M AgNO<sub>3</sub>.
 +
* All seven flasks were placed into orbital shaker at 230rpm, at 37°C, for 15 hour time period.
 +
* Absorbance on a dual-beam spectrometer was set up under photometric method, wavelength at 600nm, to measure the absorbance of each cell solution at each hour.
 +
* 3mL of LB solution was used as blank for all measurements, and 3mL of sample was taken each time for absorbance readings.
 +
 
 +
==Results==
 +
* Seven samples were run for a period of 15 hours, and absorbance at 600nm was taken at every hour.
 +
* The absorbance for each sample at each hours were shown in the following table:
 +
 
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Hour'''
 +
| align="center" style="background:#f0f0f0;"|'''Control#1 (LB+Cell)'''
 +
| align="center" style="background:#f0f0f0;"|'''Control#2 (LB+5mL AgNO3)'''
 +
| align="center" style="background:#f0f0f0;"|'''1uM AgNO3 + Cell'''
 +
| align="center" style="background:#f0f0f0;"|'''10uM AGNO3 + cell'''
 +
| align="center" style="background:#f0f0f0;"|'''100uM AGNO3 + cell'''
 +
| align="center" style="background:#f0f0f0;"|'''1mM AGNO3 + cell'''
 +
| align="center" style="background:#f0f0f0;"|'''10mM AGNO3 + cell'''
 +
|-
 +
| 0||0.113||0.366||0.116||0.122||0.148||0.178||0.387
 +
|-
 +
| 1||0.179||0.491||0.181||0.148||0.143||0.185||0.516
 +
|-
 +
| 2||0.417||0.583||0.438||0.247||0.138||0.189||0.595
 +
|-
 +
| 3||0.77||0.682||0.784||0.515||0.136||0.2||0.674
 +
|-
 +
| 4||1.296||0.783||1.258||0.925||0.134||0.201||0.754
 +
|-
 +
| 5||1.638||0.859||1.383||1.628||0.134||0.202||0.815
 +
|-
 +
| 6||1.779||0.943||1.771||1.647||0.134||0.208||0.877
 +
|-
 +
| 7||1.873||1.031||1.865||1.793||0.13||0.209||0.942
 +
|-
 +
| 8||1.929||1.098||1.933||1.872||0.132||0.213||0.987
 +
|-
 +
| 9||1.975||1.174||1.971||1.92||0.131||0.214||1.044
 +
|-
 +
| 10||2.016||1.247||2.005||1.953||0.131||0.217||1.096
 +
|-
 +
| 11||2.055||1.303||2.033||1.992||0.132||0.222||1.152
 +
|-
 +
| 12||2.06||1.37||2.06||2.008||0.132||0.225||1.201
 +
|-
 +
| 13||2.062||1.429||2.055||2.008||0.131||0.228||1.248
 +
|-
 +
| 14||2.068||1.486||2.052||2.007||0.128||0.23||1.3
 +
|-
 +
| 15||2.068||1.544||2.053||2.007||0.13||0.233||1.34
 +
|}
 +
* The graph Absorbance versus Cell growth varying concetration of AgNO<sub>3</sub> is plotted in graph below:
 +
[[Image:Absorbance versus Cell growth in different concentrations of AgNO3.jpg|600px]]
 +
 
 +
==Observation==
 +
* After the addition of 0.1M AgNO<sub>3</sub> into 50mL LB medium, the solution immediately turned opaque. Due to the small amount of AgNO<sub>3</sub> added into solutions containing AgNO<sub>3</sub> with the following concentration: 1uM, 10uM, 100uM, 1mM, and due to the limit of LB medium present, no controls were set for these samples.
 +
* The solution containing cells and 10mM AgNO<sub>3</sub> appeared to be the most opaque, a control that contained 45mL LB and 5mL 0.1M AgNO<sub>3</sub> were also run at the same time.
 +
* The opaque color of solution containing cells 10mM AgNO<sub>3</sub>, as well as the control containing 45mL LB and 5mL of 0.1M AgNO<sub>3</sub>, turned darker over time, eventually, at the point of 15 hours, the color of solution turned darkish gray.
 +
* The opaque color might be due to silver ions in solution being oxidized by the chloride present in LB media, turning a darker color.  

Current revision

Experimental Biological Chemistry II Main project page
Previous entry      Next entry

Purpose

  • Run pilot study on cell growth with concentrations of AgNO3 in solution varying from 1uM to 10mM

Procedure

  • DH5α-T1 cell pellets were taken out from -80°C freezer and left in room temperature to defrost.
  • Under a sterile environment, 3mL of autoclaved LB medium were added onto pellet. The solution was pipetted up and down gently to mix.
  • In five bottles of 125mL Erlenmeyer flasks containing 50mL LB medium, the following amount of LB were taken out of the medium from each flask: 
  4.76uL, 5.00uL, 50.0uL, 500uL, 5000uL
  • Respectively, the following amount and concentrations of AgNO3 were placed in:
Volume of LB taken out(uL) Volume of AgNO3 added(uL) Concentration of AgNO3 added(M) Final Concentration of AgNO3 in flask(uM)
4.764.760.01051
550.110
50500.1100
5005000.11,000
500050000.110,000
  • Then, 1mL of resuspended cells were placed into each of the five flasks.
  • The positive control contains 50mL of LB media and 1mL of cells
  • One more control was set up containing 45mL of LB media and 5mL of 0.1M AgNO3.
  • All seven flasks were placed into orbital shaker at 230rpm, at 37°C, for 15 hour time period.
  • Absorbance on a dual-beam spectrometer was set up under photometric method, wavelength at 600nm, to measure the absorbance of each cell solution at each hour.
  • 3mL of LB solution was used as blank for all measurements, and 3mL of sample was taken each time for absorbance readings.

Results

  • Seven samples were run for a period of 15 hours, and absorbance at 600nm was taken at every hour.
  • The absorbance for each sample at each hours were shown in the following table:
Hour Control#1 (LB+Cell) Control#2 (LB+5mL AgNO3) 1uM AgNO3 + Cell 10uM AGNO3 + cell 100uM AGNO3 + cell 1mM AGNO3 + cell 10mM AGNO3 + cell
00.1130.3660.1160.1220.1480.1780.387
10.1790.4910.1810.1480.1430.1850.516
20.4170.5830.4380.2470.1380.1890.595
30.770.6820.7840.5150.1360.20.674
41.2960.7831.2580.9250.1340.2010.754
51.6380.8591.3831.6280.1340.2020.815
61.7790.9431.7711.6470.1340.2080.877
71.8731.0311.8651.7930.130.2090.942
81.9291.0981.9331.8720.1320.2130.987
91.9751.1741.9711.920.1310.2141.044
102.0161.2472.0051.9530.1310.2171.096
112.0551.3032.0331.9920.1320.2221.152
122.061.372.062.0080.1320.2251.201
132.0621.4292.0552.0080.1310.2281.248
142.0681.4862.0522.0070.1280.231.3
152.0681.5442.0532.0070.130.2331.34
  • The graph Absorbance versus Cell growth varying concetration of AgNO3 is plotted in graph below:

Observation

  • After the addition of 0.1M AgNO3 into 50mL LB medium, the solution immediately turned opaque. Due to the small amount of AgNO3 added into solutions containing AgNO3 with the following concentration: 1uM, 10uM, 100uM, 1mM, and due to the limit of LB medium present, no controls were set for these samples.
  • The solution containing cells and 10mM AgNO3 appeared to be the most opaque, a control that contained 45mL LB and 5mL 0.1M AgNO3 were also run at the same time.
  • The opaque color of solution containing cells 10mM AgNO3, as well as the control containing 45mL LB and 5mL of 0.1M AgNO3, turned darker over time, eventually, at the point of 15 hours, the color of solution turned darkish gray.
  • The opaque color might be due to silver ions in solution being oxidized by the chloride present in LB media, turning a darker color.



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