iGEM 2012 — Team JUIT-India

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# Team:JUIT-India/Notebook

### From 2012.igem.org

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* [Home](https://2012.igem.org/Team%3AJUIT-India)
* Team
  + [Team](https://2012.igem.org/Team%3AJUIT-India/Team)
  + [Sponsors](https://2012.igem.org/Team%3AJUIT-India/Sponsors)
* Project
  + [Overview](https://2012.igem.org/Team%3AJUIT-India/Project)
  + [Protocol](https://2012.igem.org/Team%3AJUIT-India/Protocol)
* [Parts](https://2012.igem.org/Team%3AJUIT-India/Parts)
* [Modeling](https://2012.igem.org/Team%3AJUIT-India/Modeling)
* [Human Practices](https://2012.igem.org/Team%3AJUIT-India/Human_Practices)

- [Notebook](https://2012.igem.org/Team%3AJUIT-India/Notebook)
  * [Experimental Design](https://2012.igem.org/Team%3AJUIT-India/Experiment)
  * [Diary](https://2012.igem.org/Team%3AJUIT-India/WetLabWork)
  * [Brainstorming](https://2012.igem.org/Team%3AJUIT-India/References)
- [Safety](https://2012.igem.org/Team%3AJUIT-India/Safety)

![](https://static.igem.org/mediawiki/2012/5/5d/Diary.jpg)

---

Diary:

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| Contents   * [1 June](#June) * [2 July:](#July:) * [3 August:](#August:) * [4 September:](#September:) * [5 Sponsors](#Sponsors) |

## **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)

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