Team:JUIT-India/WetLabWork

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Diary:
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== ''' June ''' ==
 +
 
 +
• Brainstorming various ideas
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<br>
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• Decide the project to work on for this year
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 +
 +
==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>
 +
 +
 +
• <b>Week 4:</b><br>
 +
o PCR reaction for amplification of the genes to confirm their presence.<br>
 +
o Agarose Gel Electrophoresis<br>
 +
 Monday: PCR reaction for amplification of the various genes<br>
 +
 Tuesday : Agarose Gel Electrophoresis to confirm the genes.<br>
 +
 Wednesday: PCR reaction performed for the amplification of the various<br>
 +
 Thursday : Agarose Gel Electrophoresis <br>
 +
                      Results: Sharp Bands were observed.<br>
 +
 +
== Sponsors ==
Our Title Sponsor :
Our Title Sponsor :
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Revision as of 20:26, 25 September 2012

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


• Week 4:
o PCR reaction for amplification of the genes to confirm their presence.
o Agarose Gel Electrophoresis
 Monday: PCR reaction for amplification of the various genes
 Tuesday : Agarose Gel Electrophoresis to confirm the genes.
 Wednesday: PCR reaction performed for the amplification of the various
 Thursday : Agarose Gel Electrophoresis 

                      Results: Sharp Bands were observed.

Sponsors

Our Title Sponsor :


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