Team:JUIT-India/WetLabWork
From 2012.igem.org
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
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