Difference between revisions of "Protocol.html"

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 +
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 +
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 +
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 +
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 +
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 +
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 +
{margin-bottom:0in;}
 
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</style>
 
</style>
 
<![endif]--><!--[if gte mso 9]><xml>
 
<![endif]--><!--[if gte mso 9]><xml>
−
  <o:shapedefaults v:ext="edit" spidmax="1027"/>
+
  <o:shapedefaults v:ext="edit" spidmax="1026"/>
 
</xml><![endif]--><!--[if gte mso 9]><xml>
 
</xml><![endif]--><!--[if gte mso 9]><xml>
 
  <o:shapelayout v:ext="edit">
 
  <o:shapelayout v:ext="edit">
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</head>
 
</head>
  
−
<body lang=EN-US style='tab-interval:.5in'>
+
<body lang=EN-US link=blue vlink=purple style='tab-interval:.5in'>
  
 
<div class=WordSection1>
 
<div class=WordSection1>
  
 
<p class=MsoNormal align=center style='text-align:center'><span
 
<p class=MsoNormal align=center style='text-align:center'><span
−
style='font-size:56.0pt;line-height:115%'>INTER-LAB STUDY<o:p></o:p></span></p>
+
style='font-size:72.0pt;line-height:115%;font-family:Algerian'>PROToCOLS<o:p></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:14.0pt;font-family:"Calibri","sans-serif";
 +
mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;color:black;
 +
mso-themecolor:text1;mso-font-kerning:12.0pt'><span style='mso-list:Ignore'>1.<span
 +
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&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'>Protocol for Cloning
 +
Experiments</span></b><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'><o:p></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 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 style='margin:0in;margin-bottom:.0001pt'><span style='font-size:14.0pt'><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 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:
 +
12.0pt'>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:
 +
150%'><o:p></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:
 +
12.0pt'>(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 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:
 +
12.0pt'>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>
 +
 
 +
<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><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;mso-font-kerning:12.0pt'><span
 +
style='mso-list:Ignore'>3.<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'>Protein
 +
Characterization<o:p></o:p></span></b></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:
 +
242'><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 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:
 +
242'><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 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:
 +
242;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 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 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 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 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:
 +
14.0pt'><o:p></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 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 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:
 +
20.0pt'><o:p></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 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>References:<o:p></o:p></p>
 +
 
 +
<p class=MsoListParagraphCxSpFirst 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'>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:
 +
10.1039/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
 +
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</span></span></span><![endif]><span style='font-size:10.5pt;line-height:115%;
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font-family:"Open Sans","serif";color:black;background:white'>He, F. (2011).
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Bradford Protein Assay.&nbsp;<em><span style='font-family:"Open Sans","serif";
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mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi'>Bio-protocol</span></em>&nbsp;Bio101:
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e45. DOI:&nbsp;</span><a href="https://doi.org/10.21769/BioProtoc.45"><span
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Revision as of 00:01, 2 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, 3rd 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.