Team:JUIT-India/WetLabWork

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

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