Difference between revisions of "Protocol.html"

Revision as of 20:52, 1 November 2017

PROToCOLS

1. Protocol for Cloning Experiments

· Protocol for PCR Amplification, Competent Cell Preparation, and Transformation was followed as given in Molecular Cloning: A Laboratory Manual, 3^rd edition Sam brook.

· All Restriction Endonuclease used are of Thermo Fisher Scientific and T4DNA ligation Enzyme is of New England Bio labs and all reactions are set as per guidelines of the supplier.

2. Promoter Characterization

We had done in In-Silico Characterization of Promoter by validating the cis-element of the promoters by using Plant Care.

(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)

Functional validation of characterized MMV promoter via protoplast assay in tobacco had done by expertise of the plant biotechnology lab, Department of Botany D.E.I, Dayalbagh, Agra, Protocol had not been shared due to publication and patenting issues.

3. Protein Characterization

· Quantity Estimation of Protein: For quantify the expressed protein Bradford Method is used as given on the following link -(http://www.bio-protocol.org/e45).

· Quality Estimation of protein: The quality of expressed protein was done by SDS-PAGE.

· Protocol for the SDS-PAGE is followed as given on the following web-link- (http://www.assay-protocol.com/molecular-biology/electrophoresis/denaturing-page)

4. Immobilization of Cells Expressing our Protein of Interest

Cells of E. Coli Bl-21 expressing our desired protein are immobilized using the protocol given by Dhall et al., 2012.

(http://sci-hub.cc/http://pubs.rsc.org/en/content/articlelanding/2012/ay/c2ay25960e/unauth#!divAbstract

5. Efficacy Testing of Beads by treating with water samples possessing heavy metals contamination

Concentration of heavy metals in samples treated with our fabricated bio-beads (BECO) are analyzed by ICP-OES of Agilent Technology.

References:

1. Dhall P, Kumar R and Kumar A. 2012. BOD beads – a ready to use seeding material for estimation of organic load of wastewater. Anal. Methods, 2012, 4, 4101. DOI: 10.1039/c2ay25960e

2. He, F. (2011). Bradford Protein Assay. Bio-protocol Bio101: e45. DOI: 10.21769/BioProtoc.45.

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+color:black;mso-themecolor:text1;mso-font-kerning:12.0pt'>Protocol for Cloning
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+Experiments</span></b><span style='font-size:14.0pt;font-family:"Calibri","sans-serif";
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-  <td align=left valign=top><img width=250 height=300
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-  <td rowspan=2 align=left valign=top><img width=342 height=377
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-  alt="Text Box: Promoter Characterization&#13;&#10;&#13;&#10;iGEM student had taken part in In-Silico Characterization of Promoter by validating the cis-element of the promoters by using Plant Care.&#13;&#10;(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)&#13;&#10;Functional validation of characterized MMV promoter via protoplast assay in tobacco had done by expertise of plant biotechnology lab, Department of Botany D.E.I, Dayalbagh Agra, Protocol had not been share due to publication and patenting issues.&#13;&#10; &#13;&#10;"
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-</span></span><![endif]><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:.5in;
+margin-bottom:.0001pt;text-align:justify;text-indent:-.25in;line-height:150%;
+mso-list:l0 level1 lfo4'><![if !supportLists]><span style='font-size:14.0pt;
+line-height:150%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:
+Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:14.0pt;line-height:150%;
+font-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-bidi-font-family:
+"Times New Roman";mso-bidi-theme-font:minor-bidi;color:black;mso-themecolor:
+text1;mso-font-kerning:12.0pt'>Protocol for PCR Amplification, Competent Cell
+Preparation, and Transformation was followed as given in Molecular Cloning: A
+Laboratory Manual, 3<span class=SpellE><sup><span style='position:relative;
+top:-8.5pt;mso-text-raise:8.5pt'>rd</span></sup></span><span
+style='mso-spacerun:yes'> </span>edition Sam brook.</span><span
+style='font-size:14.0pt;line-height:150%'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:.5in;
+margin-bottom:.0001pt;text-align:justify;text-indent:-.25in;line-height:150%;
+mso-list:l0 level1 lfo4'><![if !supportLists]><span style='font-size:14.0pt;
+line-height:150%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:
+Symbol'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:14.0pt;line-height:150%;
+font-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-bidi-font-family:
+"Times New Roman";mso-bidi-theme-font:minor-bidi;color:black;mso-themecolor:
+text1;mso-font-kerning:12.0pt'>All Restriction Endonuclease used are of Thermo
+Fisher Scientific and T4DNA ligation Enzyme is of New England Bio labs and all
+reactions are set as per guidelines of the supplier.</span><span
+style='font-size:14.0pt;line-height:150%'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin:0in;margin-bottom:.0001pt'><span style='font-size:14.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p align=center style='margin-top:0in;margin-right:0in;margin-bottom:0in;
+margin-left:.5in;margin-bottom:.0001pt;text-align:center;text-indent:-.25in;
+mso-list:l1 level1 lfo1'><![if !supportLists]><b style='mso-bidi-font-weight:
+normal'><span style='font-size:20.0pt;font-family:"Calibri","sans-serif";
+mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;color:black;
+mso-themecolor:text1'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;
+</span></span></span></b><![endif]><b><span style='font-size:20.0pt;font-family:
+"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-bidi-font-family:
+"Times New Roman";mso-bidi-theme-font:minor-bidi;color:black;mso-themecolor:
+text1;mso-font-kerning:12.0pt'>Promoter Characterization</span></b><span
+style='font-size:20.0pt'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:150%'><span
+style='font-size:14.0pt;line-height:150%;font-family:"Calibri","sans-serif";
+mso-ascii-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";
+mso-bidi-theme-font:minor-bidi;color:black;mso-themecolor:text1;mso-font-kerning:
+.0pt'>We had done in <i>In-Silico</i> Characterization of Promoter by
+validating the <i style='mso-bidi-font-style:normal'>cis-</i>element of the
+promoters by using Plant Care.</span><span style='font-size:14.0pt;line-height:
+%'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:150%'><span
+style='font-size:14.0pt;line-height:150%;font-family:"Calibri","sans-serif";
+mso-ascii-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";
+mso-bidi-theme-font:minor-bidi;color:black;mso-themecolor:text1;mso-font-kerning:
+.0pt'>(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)</span><span
+style='font-size:14.0pt;line-height:150%'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:150%'><span
+style='font-size:14.0pt;line-height:150%;font-family:"Calibri","sans-serif";
+mso-ascii-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";
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+.0pt'>Functional validation of characterized MMV promoter via protoplast assay
+in tobacco had done by expertise of the plant biotechnology lab, Department of
+Botany D.E.I, <span class=SpellE>Dayalbagh</span>, <span class=GramE>Agra,</span>
+Protocol had not been shared due to publication and patenting issues.<o:p></o:p></span></p>
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+<p align=center style='margin-top:0in;margin-right:0in;margin-bottom:0in;
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+style='font-size:20.0pt;font-family:"Calibri","sans-serif";mso-ascii-theme-font:
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+Characterization<o:p></o:p></span></b></p>
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+<p style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:.5in;
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+mso-bidi-font-family:Symbol;color:#0D0D0D;mso-themecolor:text1;mso-themetint:
+'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:14.0pt;color:#0D0D0D;
+mso-themecolor:text1;mso-themetint:242;mso-font-kerning:12.0pt'>Quantity
+Estimation of Protein: For quantify the expressed protein Bradford Method is
+used as given on the following link </span><span class=GramE><span
+style='font-size:14.0pt;color:#0D0D0D;mso-themecolor:text1;mso-themetint:242'>-(</span></span><a
+href="http://www.bio-protocol.org/e45"><span style='font-size:14.0pt;
+color:#0D0D0D;mso-themecolor:text1;mso-themetint:242'>http://www.bio-protocol.org/e45</span></a><span
+style='font-size:14.0pt;color:#0D0D0D;mso-themecolor:text1;mso-themetint:242;
+mso-font-kerning:12.0pt'>).</span><span style='font-size:14.0pt;color:#0D0D0D;
+mso-themecolor:text1;mso-themetint:242'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:.5in;
+margin-bottom:.0001pt;text-align:justify;text-indent:-.25in;mso-list:l3 level1 lfo2'><![if !supportLists]><span
+style='font-size:14.0pt;font-family:Symbol;mso-fareast-font-family:Symbol;
+mso-bidi-font-family:Symbol;color:#0D0D0D;mso-themecolor:text1;mso-themetint:
+'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:14.0pt;color:#0D0D0D;
+mso-themecolor:text1;mso-themetint:242;mso-font-kerning:12.0pt'>Quality
+Estimation of protein:<span style='mso-spacerun:yes'>  </span>The quality of
+expressed protein was done by SDS-PAGE.</span><span style='font-size:14.0pt;
+color:#0D0D0D;mso-themecolor:text1;mso-themetint:242'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:.5in;
+margin-bottom:.0001pt;text-align:justify;text-indent:-.25in;mso-list:l3 level1 lfo2'><![if !supportLists]><span
+class=MsoHyperlink><span style='font-size:14.0pt;font-family:Symbol;mso-fareast-font-family:
+Symbol;mso-bidi-font-family:Symbol;color:#0D0D0D;mso-themecolor:text1;
+mso-themetint:242;text-decoration:none;text-underline:none'><span
+style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span></span><![endif]><span style='font-size:14.0pt;color:#0D0D0D;
+mso-themecolor:text1;mso-themetint:242;mso-font-kerning:12.0pt'>Protocol for
+the SD</span><span style='font-size:14.0pt;font-family:"Calibri","sans-serif";
+mso-ascii-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";
+mso-bidi-theme-font:minor-bidi;color:#0D0D0D;mso-themecolor:text1;mso-themetint:
+;mso-font-kerning:12.0pt'>S-PAGE is followed as given on the following
+web-link- (</span><a
+href="http://www.assay-protocol.com/molecular-biology/electrophoresis/denaturing-page"><span
+style='font-size:14.0pt;font-family:"Book Antiqua","serif";color:#0D0D0D;
+mso-themecolor:text1;mso-themetint:242'>http://www.assay-protocol.com/molecular-biology/electrophoresis/denaturing-page</span></a><span
+class=MsoHyperlink><span style='font-size:14.0pt;font-family:"Book Antiqua","serif";
+color:#0D0D0D;mso-themecolor:text1;mso-themetint:242'>)</span></span><span
+class=MsoHyperlink><span style='font-size:14.0pt;color:#0D0D0D;mso-themecolor:
+text1;mso-themetint:242;text-decoration:none;text-underline:none'><o:p></o:p></span></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:.5in;
+margin-bottom:.0001pt;text-align:justify'><span class=MsoHyperlink><span
+style='font-size:14.0pt;color:#0D0D0D;mso-themecolor:text1;mso-themetint:242;
+text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p align=center style='margin-top:0in;margin-right:0in;margin-bottom:0in;
+margin-left:.5in;margin-bottom:.0001pt;text-align:center;text-indent:-.25in;
+mso-list:l1 level1 lfo1'><![if !supportLists]><b style='mso-bidi-font-weight:
+normal'><span style='font-size:20.0pt;font-family:"Calibri","sans-serif";
+mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;color:black;
+mso-themecolor:text1'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;
+</span></span></span></b><![endif]><b><span style='font-size:20.0pt;font-family:
+"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-bidi-font-family:
+"Times New Roman";mso-bidi-theme-font:minor-bidi;color:black;mso-themecolor:
+text1;mso-font-kerning:12.0pt'>Immobilization of Cells Expressing our Protein
+of Interest</span></b><span style='font-size:20.0pt'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p align=center style='margin:0in;margin-bottom:.0001pt;text-align:center'><b><span
+style='font-size:20.0pt;font-family:"Calibri","sans-serif";mso-ascii-theme-font:
+minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;
+color:black;mso-themecolor:text1;mso-font-kerning:12.0pt'><o:p>&nbsp;</o:p></span></b></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin:0in;margin-bottom:.0001pt;text-align:justify'><span
+style='font-size:14.0pt;font-family:"Calibri","sans-serif";mso-ascii-theme-font:
+minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;
+color:black;mso-themecolor:text1;mso-font-kerning:12.0pt'>Cells of<i> E. Coli</i>
+Bl-21 expressing our desired protein are immobilized using the protocol given
+by <span class=SpellE>Dhall</span> et al., 2012.</span><span style='font-size:
+.0pt'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin:0in;margin-bottom:.0001pt;text-align:justify'><span
+style='font-size:10.0pt;font-family:"Calibri","sans-serif";mso-ascii-theme-font:
+minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;
+color:black;mso-themecolor:text1;mso-font-kerning:12.0pt'>(http://sci-hub.cc/http://pubs.rsc.org/en/content/articlelanding/2012/ay/c2ay25960e/unauth#!divAbstract</span><span
+style='font-size:10.0pt'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p align=center style='margin:0in;margin-bottom:.0001pt;text-align:center'><span
+style='font-size:20.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p align=center style='margin-top:0in;margin-right:0in;margin-bottom:0in;
+margin-left:.5in;margin-bottom:.0001pt;text-align:center;text-indent:-.25in;
+mso-list:l1 level1 lfo1'><![if !supportLists]><b style='mso-bidi-font-weight:
+normal'><span style='font-size:20.0pt;font-family:"Calibri","sans-serif";
+mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;color:black;
+mso-themecolor:text1'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;
+</span></span></span></b><![endif]><b><span style='font-size:20.0pt;font-family:
+"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-bidi-font-family:
+"Times New Roman";mso-bidi-theme-font:minor-bidi;color:black;mso-themecolor:
+text1;mso-font-kerning:12.0pt'>Efficacy Testing of Beads by treating with water
+samples possessing heavy metals contamination</span></b><span style='font-size:
+.0pt'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p style='margin:0in;margin-bottom:.0001pt'><span style='font-size:14.0pt;
+font-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-bidi-font-family:
+"Times New Roman";mso-bidi-theme-font:minor-bidi;color:black;mso-themecolor:
+text1;mso-font-kerning:12.0pt'>Concentration of heavy metals in samples treated
+with our fabricated bio-beads (BECO) are analyzed by ICP-<span class=GramE>OES<span
+style='mso-spacerun:yes'>  </span>of</span> Agilent Technology</span><span
+style='font-size:14.0pt;font-family:"Calibri","sans-serif";mso-ascii-theme-font:
+minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;
+color:white;mso-themecolor:light1;mso-font-kerning:12.0pt'>.</span><span
+style='font-size:14.0pt'><o:p></o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<p align=center style='margin:0in;margin-bottom:.0001pt;text-align:center'><span
+style='font-size:20.0pt'><o:p>&nbsp;</o:p></span></p>
-<br style='mso-ignore:vglayout' clear=ALL>
+<p class=MsoNormal>References:<o:p></o:p></p>
-<p class=MsoNormal><span style='mso-ansi-language:EN-IN;mso-fareast-language:
+<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l2 level1 lfo3'><![if !supportLists]><span
-EN-IN;mso-no-proof:yes'><!--[if gte vml 1]><v:shapetype id="_x0000_t75"
+style='mso-bidi-font-family:Calibri;mso-bidi-theme-font:minor-latin'><span
+style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span class=SpellE>Dhall</span> P, Kumar R and
+Kumar A. 2012. BOD beads – a ready to use seeding material for estimation of
+organic load of wastewater. Anal. Methods, 2012, 4, 4101. DOI:
+.1039/c2ay25960e<o:p></o:p></p>
+<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l2 level1 lfo3'><![if !supportLists]><span
+style='mso-bidi-font-family:Calibri;mso-bidi-theme-font:minor-latin'><span
+style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:10.5pt;line-height:115%;
+font-family:"Open Sans","serif";color:black;background:white'>He, F. (2011).
+Bradford Protein Assay.&nbsp;<em><span style='font-family:"Open Sans","serif";
+mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi'>Bio-protocol</span></em>&nbsp;Bio101:
+e45. DOI:&nbsp;</span><a href="https://doi.org/10.21769/BioProtoc.45"><span
+style='font-size:10.5pt;line-height:115%;font-family:"Open Sans","serif";
+color:#0094FF;background:white'>10.21769/BioProtoc.45</span></a><span
+style='font-size:10.5pt;line-height:115%;font-family:"Open Sans","serif";
+color:black;background:white'>.</span></p>
+<p class=MsoListParagraphCxSpMiddle><span style='font-size:10.5pt;line-height:
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-    <p class=MsoNormal><span lang=EN-US>References::</span></p>
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-    l0 level1 lfo1'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:
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-    style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-    lang=EN-US>Dhall P, Kumar R and Kumar A. 2012. BOD beads – a ready to use
-    seeding material for estimation of organic load of wastewater. Anal.
-    Methods, 2012, 4, 4101. DOI: 10.1039/c2ay25960e</span></p>
-    <p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt;mso-list:
-    l0 level1 lfo1'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:
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-    style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-    lang=EN-US style='font-size:10.5pt;line-height:115%;font-family:"Open Sans","serif";
-    color:black;background:white'>He, F. (2011). Bradford Protein Assay.&nbsp;<em><span
-    style='font-family:"Open Sans","serif";mso-bidi-font-family:"Times New Roman";
-    mso-bidi-theme-font:minor-bidi'>Bio-protocol</span></em>&nbsp;Bio101: e45.
-    DOI:&nbsp;</span><span lang=EN-US><a
-    href="https://doi.org/10.21769/BioProtoc.45"><span style='font-size:10.5pt;
-    line-height:115%;font-family:"Open Sans","serif";color:#0094FF;background:
-    white'>10.21769/BioProtoc.45</span></a></span><span lang=EN-US
-    style='font-size:10.5pt;line-height:115%;font-family:"Open Sans","serif";
-    color:black;background:white'>.</span></p>
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-alt="Text Box: References::&#13;&#10;1.	Dhall P, Kumar R and Kumar A. 2012. BOD beads – a ready to use seeding material for estimation of organic load of wastewater. Anal. Methods, 2012, 4, 4101. DOI: 10.1039/c2ay25960e&#13;&#10;2.	He, F. (2011). Bradford Protein Assay. Bio-protocol Bio101: e45. DOI: 10.21769/BioProtoc.45.&#13;&#10;&#13;&#10;"
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