Team:JUIT-India/Notebook
From 2012.igem.org
(Prototype team page) |
|||
(2 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
- | |||
- | |||
<html> | <html> | ||
- | < | + | <style> |
- | + | #contentSub, #search-controls, .firstHeading, #footer-box, #catlinks, #p-logo { | |
- | + | display:none;} | |
- | + | #top-section { | |
- | + | border: 0px; | |
- | + | height: 0px;} | |
- | </ | + | #content { |
- | < | + | border: none;} |
- | + | /* Removes "teams" from the menubar */ | |
- | </ | + | #menubar > ul > li:last-child { |
+ | display: none;} | ||
+ | /* Resizes the menubar to fik the links (default is 400px) */ | ||
+ | #menubar { | ||
+ | width: auto;} | ||
+ | body { | ||
+ | margin: 10px 0 0 0; | ||
+ | padding: 5;} | ||
+ | #top-section { | ||
+ | width: 965px; | ||
+ | height: 0; | ||
+ | margin: 0 auto; | ||
+ | padding: 0; | ||
+ | border: none;} | ||
+ | #menubar { | ||
+ | font-size: 65%; | ||
+ | top: -14 px;} | ||
+ | .left-menu:hover { | ||
+ | background-color: transparent;} | ||
+ | #menubar li a { | ||
+ | background-color: transparent;} | ||
+ | #menubar:hover { | ||
+ | color: white;} | ||
+ | #menubar li a { | ||
+ | color: transparent;} | ||
+ | #menubar:hover li a { | ||
+ | color: white;} | ||
+ | |||
+ | |||
+ | img | ||
+ | { | ||
+ | opacity:1.0; | ||
+ | filter:alpha(opacity=100); | ||
+ | } | ||
+ | img:hover | ||
+ | { | ||
+ | opacity:1.0; | ||
+ | filter:alpha(opacity=100); | ||
+ | } | ||
+ | .cssmenu{ | ||
+ | border:none; | ||
+ | border:0px; | ||
+ | margin:0px; | ||
+ | padding:0px; | ||
+ | font: 67.5% 'Lucida Sans Unicode', 'Bitstream Vera Sans', 'Trebuchet Unicode MS', 'Lucida Grande', Verdana, Helvetica, sans-serif; | ||
+ | font-size:14px; | ||
+ | font-weight:bold; | ||
+ | } | ||
+ | .cssmenu ul{ | ||
+ | background:#333000; | ||
+ | height:35px; | ||
+ | list-style:none; | ||
+ | margin:0; | ||
+ | padding:0; | ||
+ | } | ||
+ | .cssmenu li{ | ||
+ | float:left; | ||
+ | padding: 0px; | ||
+ | } | ||
+ | .cssmenu li a{ | ||
+ | background:#333333 url('https://static.igem.org/mediawiki/2012/3/3f/Seperator.gif') bottom right no-repeat; | ||
+ | color:#cccccc; | ||
+ | display:block; | ||
+ | font-weight:normal; | ||
+ | line-height:35px; | ||
+ | margin:0px; | ||
+ | padding:0px 25px; | ||
+ | text-align:center; | ||
+ | text-decoration:none; | ||
+ | } | ||
+ | .cssmenu li a:hover, .cssmenu ul li:hover a{ | ||
+ | background: #2580a2 url('https://static.igem.org/mediawiki/2012/e/e6/Hover.gif') bottom center no-repeat; | ||
+ | |||
+ | text-decoration:none; | ||
+ | } | ||
+ | .cssmenu li ul{ | ||
+ | background:#333333; | ||
+ | display:none; | ||
+ | height:auto; | ||
+ | padding:0px; | ||
+ | margin:0px; | ||
+ | border:0px; | ||
+ | position:absolute; | ||
+ | width:225px; | ||
+ | z-index:200; | ||
+ | /*top:1em; | ||
+ | /*left:0;*/ | ||
+ | } | ||
+ | .cssmenu li:hover ul{ | ||
+ | display:block; | ||
+ | |||
+ | } | ||
+ | .cssmenu li li { | ||
+ | background:url('https://static.igem.org/mediawiki/2012/3/3f/Sub_sep.gif') bottom left no-repeat; | ||
+ | display:block; | ||
+ | float:none; | ||
+ | margin:0px; | ||
+ | padding:0px; | ||
+ | width:225px; | ||
+ | } | ||
+ | .cssmenu li:hover li a{ | ||
+ | background:none; | ||
+ | |||
+ | } | ||
+ | .cssmenu li ul a{ | ||
+ | display:block; | ||
+ | height:35px; | ||
+ | font-size:12px; | ||
+ | font-style:normal; | ||
+ | margin:0px; | ||
+ | padding:0px 10px 0px 15px; | ||
+ | text-align:left; | ||
+ | } | ||
+ | .cssmenu li ul a:hover, .cssmenu li ul li:hover a{ | ||
+ | background:#2580a2 url('https://static.igem.org/mediawiki/2012/a/a8/Hover_sub.gif') center left no-repeat; | ||
+ | border:0px; | ||
+ | |||
+ | text-decoration:none; | ||
+ | } | ||
+ | .cssmenu p{ | ||
+ | clear:left; | ||
+ | } | ||
+ | </style> | ||
+ | <div class='cssmenu' z-index:5000> | ||
+ | <div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div> | ||
+ | <ul> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li> | ||
+ | <li><a href='#'><span>Team</span></a> | ||
+ | <ul> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li><a href='#'><span>Project</span></a> | ||
+ | <ul> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> | ||
+ | |||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a> | ||
+ | <ul> | ||
+ | </a></li> | ||
+ | |||
+ | </ul> | ||
+ | </li> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a> | ||
+ | <ul> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li> | ||
+ | |||
+ | </ul> | ||
+ | </li> | ||
+ | <li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li> | ||
+ | |||
+ | </ul> | ||
</div> | </div> | ||
+ | <div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/5d/Diary.jpg" width="965" height="300"></div> | ||
+ | |||
+ | <r> | ||
+ | <hr> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
</html> | </html> | ||
+ | Diary: | ||
- | + | == ''' June ''' == | |
- | + | • Brainstorming various ideas | |
- | + | <br> | |
- | + | • Decide the project to work on for this year | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
+ | |||
+ | ==July:== | ||
+ | <br>• Week 1 | ||
+ | <br>o Research on the project thoroughly | ||
+ | <br>o Primers Designing | ||
+ | • Week 2 | ||
+ | <br>o Culturing of M.Capsulatus | ||
+ | <br>o Isolation of Genomic DNA from M.Capsulatus | ||
+ | <br>o PCR reaction for amplification of MxaF | ||
+ | <br>o Agarose Gel Electrophoresis | ||
+ | <br>o Optimization & Standardization of PCR | ||
+ | <br> Monday : Cultured M.Capsulatus | ||
+ | <br> Tuesday : Isolated Genomic DNA from M.Capsulatus | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br> Wednesday : PCR Reaction for amplification of MxaF | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | Result: Non-Specific Binding<br> | ||
+ | Thursday : PCR reaction performed again | ||
+ | <br>Agarose Gel Electrophoresis<br> | ||
+ | Result: NO bands were observed | ||
+ | <br> Friday : PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis<br> | ||
+ | Result: NO bands were observed | ||
+ | <br> Saturday: PCR reaction performed | ||
+ | Agarose Gel Electrophoresis<br> | ||
+ | Result: light bands were observed | ||
+ | <br> Sunday: PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: bands were observed | ||
+ | <br>Gel Extraction. | ||
+ | |||
+ | |||
+ | <br>• Week 3 | ||
+ | <br>o Culturing of M.Capsulatus | ||
+ | <br>o Isolation of Genomic DNA from M.Capsulatus | ||
+ | <br>o PCR reaction for amplification of NifA | ||
+ | <br>o Agarose Gel Electrophoresis | ||
+ | <br>o Optimization & Standardization of PCR | ||
+ | <br> Monday : Cultured M.Capsulatus | ||
+ | <br> Tuesday : Isolated Genomic DNA from M.Capsulatus | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Results: NO band was observed (due to less duration in the -200c freezer | ||
+ | <br> Wednesday : Isolated Genomic DNA from M.Capsulatus | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: Sharp Bands were observed | ||
+ | <br> Thursday : PCR reaction performed for amplification of NifA | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: NO bands were observed | ||
+ | <br> Friday : PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: Nonspecific bands were observed | ||
+ | <br> Saturday: PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: Nonspecific bands were observed | ||
+ | <br> Sunday: PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: Sharp bands were observed | ||
+ | <br>Gel Extraction. | ||
+ | |||
+ | <br>• Week 4 | ||
+ | <br>o Culturing of B.subtilis | ||
+ | <br>o Isolation of genomic DNA from B.subtilis | ||
+ | <br>o PCR reaction for amplification of SacB | ||
+ | <br>o Agarose Gel Electrophoresis | ||
+ | <br>o Optimization & Standardization of PCR | ||
+ | <br> Monday : Cultured B.subtilis | ||
+ | <br> Tuesday : Isolated Genomic DNA from B.subtilis | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br> Wednesday : PCR Reaction for amplification of SacB | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: Non-Specific Binding | ||
+ | <br> Thursday : PCR reaction performed again | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: NO bands were observed | ||
+ | <br> Friday : PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: NO bands were observed | ||
+ | <br> Saturday: PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: light bands were observed | ||
+ | <br> Sunday: PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | <br>Result: bands were observed | ||
+ | <br>Gel Extraction was performed. | ||
+ | |||
+ | |||
+ | ==August: == | ||
+ | <br>• Week 1 | ||
+ | <br>o Culturing of P. aeruginosa | ||
+ | <br>o Isolation of genomic DNA from P. aeruginosa | ||
+ | <br>o PCR Reaction for amplification of NosZ | ||
+ | <br>o Agarose Gel Electrophoresis | ||
+ | <br>o Optimization & Standardization of PCR | ||
+ | <br> Monday : Cultured P. aeruginosa | ||
+ | <br> Tuesday : Isolated Genomic DNA from P. aeruginosa | ||
+ | Agarose Gel Electrophoresis | ||
+ | Results: NO band was observed | ||
+ | <br> Wednesday : Isolated Genomic DNA from P. aeruginosa using CTAB method | ||
+ | Agarose Gel Electrophoresis | ||
+ | Result: Sharp Bands were observed | ||
+ | <br> Thursday : PCR reaction performed for amplification of NosZ | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | Result: NO bands were observed | ||
+ | <br> Friday : PCR reaction performed | ||
+ | Agarose Gel Electrophoresis | ||
+ | Result: No bands were observed | ||
+ | <br> Saturday: PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | Result: Nonspecific bands were observed | ||
+ | <br> Sunday: PCR reaction performed | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | Result: Sharp bands were observed | ||
+ | <br>Gel Extraction was performed. | ||
+ | |||
+ | |||
+ | |||
+ | <br>• Week 2 | ||
+ | <br>o PCR reactions for amplification of MxaF & NifA | ||
+ | <br>o Gel Extraction for the genes | ||
+ | <br>o Ligation of genes into TA Vector | ||
+ | <br>o Transformation of E.Coli cells with TA Vector | ||
+ | <br>o Culturing of Transformed Cells – Starter Culture | ||
+ | <br> Monday : PCR reaction performed for MxaF & NifA | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | Result: Sharp bands were observed | ||
+ | <br>Gel Extraction was performed. | ||
+ | <br> Tuesday : Ligation of MxaF into TA Vector | ||
+ | <br> Wednesday : Transformation using MgCl2 & CaCl2 method | ||
+ | Result: Failed(Contamination) | ||
+ | <br> Thursday : Ligation of MxaF into TA Vector | ||
+ | <br> Friday: Transformation using MgCl2 & CaCl2 method | ||
+ | Result: White Colonies were selected and cultured | ||
+ | <br> Saturday: Ligation of NifA into TA Vector | ||
+ | <br> Sunday: : Transformation using MgCl2 & CaCl2 method | ||
+ | Result: White Colonies were selected and cultured | ||
+ | o | ||
+ | |||
+ | <br>• Week 3 | ||
+ | <br>o PCR reactions for amplification of NosZ & SacB | ||
+ | <br>o Gel Extraction for the genes | ||
+ | <br>o Ligation of genes into TA Vector | ||
+ | <br>o Transformation of E.Coli cells with TA Vector | ||
+ | <br>o Culturing of Transformed Cells – Starter Culture\ | ||
+ | <br> Monday : PCR reaction performed for NosZ & SacB | ||
+ | <br>Agarose Gel Electrophoresis | ||
+ | Result: Sharp bands were observed | ||
+ | <br>Gel Extraction was performed. | ||
+ | <br> Tuesday : Ligation of NosZ into TA Vector<br> | ||
+ | Wednesday : Transformation using MgCl2 & CaCl2 method | ||
+ | Result: White Colonies were selected and cultured | ||
+ | Thursday : Ligation of SacB into TA Vector<br> | ||
+ | Friday: Transformation using MgCl2 & CaCl2 method<br> | ||
+ | Result: No colonies were observed<br> | ||
+ | Saturday: Ligation of SacB into TA Vector <br> | ||
+ | Sunday: : Transformation using MgCl2 & CaCl2 method<br> | ||
+ | Result: White Colonies were selected and cultured<br> | ||
+ | |||
+ | • Week 4<br> | ||
+ | o Culture transformed Cells<br> | ||
+ | o Plasmid Isolation<br> | ||
+ | o PCR reactions to confirm the insertion of genes<br> | ||
+ | o Agarose Gel Electrophoresis<br> | ||
+ | Monday : Cultured MxaF, NifA, SacB & NosZ transformed Cells<br> | ||
+ | Tuesday : Plasmid Isolation for MxaF & NifA transformed cells | ||
+ | Restriction Digestion of the plasmids<br> | ||
+ | Wednesday : PCR reaction performed for amplification of MxaF & NifA | ||
+ | Agarose Gel Electrophoresis. <br> | ||
+ | Results: Sharp Bands were observed.<br> | ||
+ | Thursday : Plasmid Isolation for NosZ & SacB transformed cells | ||
+ | Restriction Digestion of the plasmids.<br> | ||
+ | Agarose Gel Electrophoresis<br> | ||
+ | Friday: PCR reaction performed for amplification of MxaF & NifA <br> | ||
+ | Saturday: Agarose Gel Electrophoresis<br> | ||
+ | |||
+ | |||
+ | ==September: == | ||
+ | • Week 1<br> | ||
+ | o Restriction Digestion of MxaF containing plasmids<br> | ||
+ | o Ligation of MxaF into psb1c3<br> | ||
+ | o Restriction Digestion of NosZcontaining plasmids<br> | ||
+ | o Ligation of NosZ into psb1c3 containg MxaF<br> | ||
+ | Monday : Restriction Digestion of MxaF containing plasmids<br> | ||
+ | Agarose Gel Electrophoresis<br> | ||
+ | Result: Sharp bands were observed<br> | ||
+ | Gel Extraction was performed.<br> | ||
+ | Tuesday : Restriction Digestion of psb1c3 using EcoR1 | ||
+ | Ligation of MxaF into psb1c3<br> | ||
+ | Thursday : Restriction Digestion of NosZ containing plasmids<br> | ||
+ | Agarose Gel Electrophoresis<br> | ||
+ | Result: Sharp bands were observed<br> | ||
+ | Friday: Transformation using MgCl2 & CaCl2 method <br> | ||
+ | Saturday: Restriction Digestion of psb1c3<br> | ||
+ | Ligation of NosZ into psb1c3 containing MxaF<br> | ||
+ | |||
+ | • <b>Week 2:</b><br> | ||
+ | o Restriction Digestion of NifA<br> | ||
+ | o Ligation of NifA into psb1c3 containg NifA & SacB<br> | ||
+ | o Restriction Digestion of SacB<br> | ||
+ | o Ligation of SacB into psb1c3 containg all the other genes<br> | ||
+ | Monday : Restriction Digestion of NifA containing plasmids<br> | ||
+ | Agarose Gel Electrophoresis<br> | ||
+ | Result: Sharp bands were observed<br> | ||
+ | Gel Extraction was performed.<br> | ||
+ | Tuesday : Restriction Digestion of psb1c3 containing MxaF & NosZ using EcoR1 | ||
+ | Ligation of NifA into psb1c3<br> | ||
+ | Thursday : Restriction Digestion of SacB containing plasmids<br> | ||
+ | Agarose Gel Electrophoresis<br> | ||
+ | Result: Sharp bands were observed<br> | ||
+ | Friday: Transformation using MgCl2 & CaCl2 method <br> | ||
+ | Saturday: Restriction Digestion of psb1c3<br> | ||
+ | Ligation of SacB into psb1c3 containing all the other genes.<br> | ||
+ | |||
+ | • <b>Week 3:</b><br> | ||
+ | o Transformation of M.Capsulatus competent Cells using the plasmid containing all the genes<br> | ||
+ | o Growth of transformed cells on selective media.<br> | ||
+ | o Plasmid Isolation <br> | ||
+ | o PCR reaction to confirm the insertion of genes<br> | ||
+ | Monday: Cultures M.Capsulatus Cells<br> | ||
+ | Tuesday : Preparation of M.Capsulatus Competent Cells<br> | ||
+ | Wednesday: Transformation of competent cells using the prepared plasmids<br> | ||
+ | Thursday : Growth of plates on LB plates containg chloramphenicol<br> | ||
+ | Friday: Cultured transformed cells in LB agar<br> | ||
+ | Saturday: Plasmid Isolation <br> | ||
+ | Restriction Digestion<br> | ||
+ | Sunday: Agarose Gel Electrophoresis<br> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | == Sponsors == | ||
+ | Our Title Sponsor : | ||
+ | <html> | ||
+ | <br> | ||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" > | ||
+ | </center> | ||
+ | </html> | ||
- | + | {|align="justify" |
Latest revision as of 18:51, 26 September 2012
![](https://static.igem.org/mediawiki/2012/5/5d/Diary.jpg)
Diary:
Contents |
June
• Brainstorming various ideas
• Decide the project to work on for this year
July:
• Week 1
o Research on the project thoroughly
o Primers Designing
• Week 2
o Culturing of M.Capsulatus
o Isolation of Genomic DNA from M.Capsulatus
o PCR reaction for amplification of MxaF
o Agarose Gel Electrophoresis
o Optimization & Standardization of PCR
Monday : Cultured M.Capsulatus
Tuesday : Isolated Genomic DNA from M.Capsulatus
Agarose Gel Electrophoresis
Wednesday : PCR Reaction for amplification of MxaF
Agarose Gel Electrophoresis
Result: Non-Specific Binding
Thursday : PCR reaction performed again
Agarose Gel Electrophoresis
Result: NO bands were observed
Friday : PCR reaction performed
Agarose Gel Electrophoresis
Result: NO bands were observed
Saturday: PCR reaction performed
Agarose Gel Electrophoresis
Result: light bands were observed
Sunday: PCR reaction performed
Agarose Gel Electrophoresis
Result: bands were observed
Gel Extraction.
• Week 3
o Culturing of M.Capsulatus
o Isolation of Genomic DNA from M.Capsulatus
o PCR reaction for amplification of NifA
o Agarose Gel Electrophoresis
o Optimization & Standardization of PCR
Monday : Cultured M.Capsulatus
Tuesday : Isolated Genomic DNA from M.Capsulatus
Agarose Gel Electrophoresis
Results: NO band was observed (due to less duration in the -200c freezer
Wednesday : Isolated Genomic DNA from M.Capsulatus
Agarose Gel Electrophoresis
Result: Sharp Bands were observed
Thursday : PCR reaction performed for amplification of NifA
Agarose Gel Electrophoresis
Result: NO bands were observed
Friday : PCR reaction performed
Agarose Gel Electrophoresis
Result: Nonspecific bands were observed
Saturday: PCR reaction performed
Agarose Gel Electrophoresis
Result: Nonspecific bands were observed
Sunday: PCR reaction performed
Agarose Gel Electrophoresis
Result: Sharp bands were observed
Gel Extraction.
• Week 4
o Culturing of B.subtilis
o Isolation of genomic DNA from B.subtilis
o PCR reaction for amplification of SacB
o Agarose Gel Electrophoresis
o Optimization & Standardization of PCR
Monday : Cultured B.subtilis
Tuesday : Isolated Genomic DNA from B.subtilis
Agarose Gel Electrophoresis
Wednesday : PCR Reaction for amplification of SacB
Agarose Gel Electrophoresis
Result: Non-Specific Binding
Thursday : PCR reaction performed again
Agarose Gel Electrophoresis
Result: NO bands were observed
Friday : PCR reaction performed
Agarose Gel Electrophoresis
Result: NO bands were observed
Saturday: PCR reaction performed
Agarose Gel Electrophoresis
Result: light bands were observed
Sunday: PCR reaction performed
Agarose Gel Electrophoresis
Result: bands were observed
Gel Extraction was performed.
August:
• Week 1
o Culturing of P. aeruginosa
o Isolation of genomic DNA from P. aeruginosa
o PCR Reaction for amplification of NosZ
o Agarose Gel Electrophoresis
o Optimization & Standardization of PCR
Monday : Cultured P. aeruginosa
Tuesday : Isolated Genomic DNA from P. aeruginosa
Agarose Gel Electrophoresis
Results: NO band was observed
Wednesday : Isolated Genomic DNA from P. aeruginosa using CTAB method
Agarose Gel Electrophoresis
Result: Sharp Bands were observed
Thursday : PCR reaction performed for amplification of NosZ
Agarose Gel Electrophoresis
Result: NO bands were observed
Friday : PCR reaction performed
Agarose Gel Electrophoresis
Result: No bands were observed
Saturday: PCR reaction performed
Agarose Gel Electrophoresis
Result: Nonspecific bands were observed
Sunday: PCR reaction performed
Agarose Gel Electrophoresis
Result: Sharp bands were observed
Gel Extraction was performed.
• Week 2
o PCR reactions for amplification of MxaF & NifA
o Gel Extraction for the genes
o Ligation of genes into TA Vector
o Transformation of E.Coli cells with TA Vector
o Culturing of Transformed Cells – Starter Culture
Monday : PCR reaction performed for MxaF & NifA
Agarose Gel Electrophoresis
Result: Sharp bands were observed
Gel Extraction was performed.
Tuesday : Ligation of MxaF into TA Vector
Wednesday : Transformation using MgCl2 & CaCl2 method
Result: Failed(Contamination)
Thursday : Ligation of MxaF into TA Vector
Friday: Transformation using MgCl2 & CaCl2 method
Result: White Colonies were selected and cultured
Saturday: Ligation of NifA into TA Vector
Sunday: : Transformation using MgCl2 & CaCl2 method
Result: White Colonies were selected and cultured
o
• Week 3
o PCR reactions for amplification of NosZ & SacB
o Gel Extraction for the genes
o Ligation of genes into TA Vector
o Transformation of E.Coli cells with TA Vector
o Culturing of Transformed Cells – Starter Culture\
Monday : PCR reaction performed for NosZ & SacB
Agarose Gel Electrophoresis
Result: Sharp bands were observed
Gel Extraction was performed.
Tuesday : Ligation of NosZ into TA Vector
Wednesday : Transformation using MgCl2 & CaCl2 method
Result: White Colonies were selected and cultured
Thursday : Ligation of SacB into TA Vector
Friday: Transformation using MgCl2 & CaCl2 method
Result: No colonies were observed
Saturday: Ligation of SacB into TA Vector
Sunday: : Transformation using MgCl2 & CaCl2 method
Result: White Colonies were selected and cultured
• Week 4
o Culture transformed Cells
o Plasmid Isolation
o PCR reactions to confirm the insertion of genes
o Agarose Gel Electrophoresis
Monday : Cultured MxaF, NifA, SacB & NosZ transformed Cells
Tuesday : Plasmid Isolation for MxaF & NifA transformed cells
Restriction Digestion of the plasmids
Wednesday : PCR reaction performed for amplification of MxaF & NifA
Agarose Gel Electrophoresis.
Results: Sharp Bands were observed.
Thursday : Plasmid Isolation for NosZ & SacB transformed cells
Restriction Digestion of the plasmids.
Agarose Gel Electrophoresis
Friday: PCR reaction performed for amplification of MxaF & NifA
Saturday: Agarose Gel Electrophoresis
September:
• Week 1
o Restriction Digestion of MxaF containing plasmids
o Ligation of MxaF into psb1c3
o Restriction Digestion of NosZcontaining plasmids
o Ligation of NosZ into psb1c3 containg MxaF
Monday : Restriction Digestion of MxaF containing plasmids
Agarose Gel Electrophoresis
Result: Sharp bands were observed
Gel Extraction was performed.
Tuesday : Restriction Digestion of psb1c3 using EcoR1
Ligation of MxaF into psb1c3
Thursday : Restriction Digestion of NosZ containing plasmids
Agarose Gel Electrophoresis
Result: Sharp bands were observed
Friday: Transformation using MgCl2 & CaCl2 method
Saturday: Restriction Digestion of psb1c3
Ligation of NosZ into psb1c3 containing MxaF
• Week 2:
o Restriction Digestion of NifA
o Ligation of NifA into psb1c3 containg NifA & SacB
o Restriction Digestion of SacB
o Ligation of SacB into psb1c3 containg all the other genes
Monday : Restriction Digestion of NifA containing plasmids
Agarose Gel Electrophoresis
Result: Sharp bands were observed
Gel Extraction was performed.
Tuesday : Restriction Digestion of psb1c3 containing MxaF & NosZ using EcoR1
Ligation of NifA into psb1c3
Thursday : Restriction Digestion of SacB containing plasmids
Agarose Gel Electrophoresis
Result: Sharp bands were observed
Friday: Transformation using MgCl2 & CaCl2 method
Saturday: Restriction Digestion of psb1c3
Ligation of SacB into psb1c3 containing all the other genes.
• Week 3:
o Transformation of M.Capsulatus competent Cells using the plasmid containing all the genes
o Growth of transformed cells on selective media.
o Plasmid Isolation
o PCR reaction to confirm the insertion of genes
Monday: Cultures M.Capsulatus Cells
Tuesday : Preparation of M.Capsulatus Competent Cells
Wednesday: Transformation of competent cells using the prepared plasmids
Thursday : Growth of plates on LB plates containg chloramphenicol
Friday: Cultured transformed cells in LB agar
Saturday: Plasmid Isolation
Restriction Digestion
Sunday: Agarose Gel Electrophoresis
Sponsors
Our Title Sponsor :
![](https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg)