Team:RMIT Australia/Notebook

From 2010.igem.org

(Difference between revisions)
(→Notebook)
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<div id=" .2F21_May_2010"></div>
<div id=" .2F21_May_2010"></div>
-
<div id=" .2F28_June_2010">'''28 June'''
+
<div id=" .2F28_June_2010">
 +
===28 June===
Did OH&S training and PC2 training
Did OH&S training and PC2 training
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</div>
</div>
-
<div id=" .2F29_June_2010">'''29 June'''
+
<div id=" .2F29_June_2010">
 +
===29 June===
Began research on what we were going to use to promote taq
Began research on what we were going to use to promote taq
</div>
</div>
-
<div id=" .2F30_June_2010">'''30 June'''
+
<div id=" .2F30_June_2010">
 +
===30 June===
Looked at T7 with a Lac operon as a promotore of taq  
Looked at T7 with a Lac operon as a promotore of taq  
</div>
</div>
-
<div id=" .2F13_July_2010">'''13 July'''
+
<div id=" .2F13_July_2010">
 +
===13 July===
Designed Primers for Taq into backbone
Designed Primers for Taq into backbone
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</div>
</div>
-
<div id=" .2F14_July_2010">'''14 July'''
+
<div id=" .2F14_July_2010">
 +
===14 July===
Looked at prices of Hotels in Boston
Looked at prices of Hotels in Boston
</div>
</div>
-
<div id=" .2F19_July_2010">'''19 July'''
+
<div id=" .2F19_July_2010">
 +
===19 July===
began looking at the structure of taq, its thermostability
began looking at the structure of taq, its thermostability
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</div>
</div>
-
<div id=" .2F21_July_2010">'''21 July'''
+
<div id=" .2F21_July_2010">
 +
===21 July===
sent in sequence of wild type taq to iTasser
sent in sequence of wild type taq to iTasser
</div>
</div>
-
<div id=" .2F23_July_2010">'''23 July'''
+
<div id=" .2F23_July_2010">
 +
===23 July===
Checked thermostability of the mutations that will be done to Taq using Expasy
Checked thermostability of the mutations that will be done to Taq using Expasy
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Sent in sequence of taq with mutations at 485, 515 and 540 to iTasser
Sent in sequence of taq with mutations at 485, 515 and 540 to iTasser
   
   
-
<div id=" .2F3_August_2010">'''3 August'''
+
<div id=" .2F3_August_2010">
 +
===3 August===
Had iGEM team meeting
Had iGEM team meeting
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</div>
</div>
-
<div id=" .2F5_August_2010">'''5 August'''
+
<div id=" .2F5_August_2010">
 +
===5 August===
Meet up with Damian, discussed different strategies to use in modelling, began using YASARA to do our Modelling
Meet up with Damian, discussed different strategies to use in modelling, began using YASARA to do our Modelling
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</div>
</div>
-
<div id=" .2F10_August_2010">'''10 August'''
+
<div id=" .2F10_August_2010">
 +
===10 August===
Had iGEM team meeting
Had iGEM team meeting
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</div>
</div>
-
<div id=" .2F11_August_2010">'''11 August'''
+
<div id=" .2F11_August_2010">===11 August===
PCR of Taq
PCR of Taq
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</div>
</div>
-
<div id=" .2F16_August_2010">'''16 August'''
+
<div id=" .2F16_August_2010">
 +
===16 August===
Collaborated with the Sheffield team by participating in an over the phone survery about Human Practices
Collaborated with the Sheffield team by participating in an over the phone survery about Human Practices
-
<div id=" .2F17_August_2010">'''17 August'''
 
</div>
</div>
 +
<div id=" .2F17_August_2010">
 +
===17 August===
 +
had team meeting  
had team meeting  
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</div>
</div>
-
<div id=" .2F18_August_2010">'''18 August'''
+
<div id=" .2F18_August_2010">
 +
===18 August===
called few companies seeking sponsorship
called few companies seeking sponsorship
enquired for booking flights and accomadation
enquired for booking flights and accomadation
-
</div id=" .2F19_August_2010">'''19 August'''
+
</div id=" .2F19_August_2010">
 +
===19 August===
Working on comparing TAQ wild structure to the TAQ Mutated structure to see that there are no major differences, which will mean that TAQ is inactive but will retain it's thermostability at high temperatures.  
Working on comparing TAQ wild structure to the TAQ Mutated structure to see that there are no major differences, which will mean that TAQ is inactive but will retain it's thermostability at high temperatures.  
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|}
|}
-
<div id=" .2F20_August_2010">'''20 August'''
+
<div id=" .2F20_August_2010">
 +
===20 August===
 +
 
Did two PCR reaction and a transformation
Did two PCR reaction and a transformation
The first PCR we ran was a touch down PCR. Temperatures ranged from 69 to 64.
The first PCR we ran was a touch down PCR. Temperatures ranged from 69 to 64.
The second PCR was a two step PCR.
The second PCR was a two step PCR.
-
<div id=" .2F30_August_2010">'''30 August'''
+
<div id=" .2F30_August_2010">
 +
===30 August===
Ordered the TAQ polymerase from Mr. Gene. Should have it in 15 days.
Ordered the TAQ polymerase from Mr. Gene. Should have it in 15 days.
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After the meeting we all are sitting together for a writing session.
After the meeting we all are sitting together for a writing session.
-
<div id=" .2F31_August_2010">'''31 August'''
+
<div id=" .2F31_August_2010">
 +
===31 August===
Spent the morning organising our flights
Spent the morning organising our flights
In the afternoon we all sat together for a writing session
In the afternoon we all sat together for a writing session
-
<div id=" .2F1_September_2010">'''1 September'''
+
<div id=" .2F1_September_2010">
 +
===1 September===
We all sat together for another writing session.
We all sat together for another writing session.
-
<div id=" .2F7_September_2010">'''7 September'''
+
<div id=" .2F7_September_2010">
 +
===7 September===
Had a team meeting
Had a team meeting
-
<div id=" .2F14_September_2010">'''14 September'''
+
<div id=" .2F14_September_2010">
 +
===14 September===
Had a team meeting and made a lab time table, we also set out tasks which need to be done by the next meeting  
Had a team meeting and made a lab time table, we also set out tasks which need to be done by the next meeting  
-
- Update the wiki
+
*Update the wiki
-
- Finalise the form for the ethics committee for pennstate
+
* Finalise the form for the ethics committee for pennstate
-
- Call up possible sponsors
+
* Call up possible sponsors
-
- Collect Tshirts
+
* Collect Tshirts
-
- Work on abstract and team Roster
+
* Work on abstract and team Roster
-
- research heat liable bonds
+
* research heat liable bonds
-
- order peptides
+
* order peptides
-
- chat with emilio about possible bio assays
+
* chat with emilio about possible bio assays
-
- work on making the promotor part
+
* work on making the promotor part
-
<div id=" .2F15_September_2010">'''15 September'''
+
<div id=" .2F15_September_2010">
 +
===15 September===
'''Mutagenesis Quikchange XL kit'''
'''Mutagenesis Quikchange XL kit'''
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Dpn1 digest (2 µl) for 2hrs
Dpn1 digest (2 µl) for 2hrs
-
<div id=" .2F16_September_2010">'''16 September'''
+
<div id=" .2F16_September_2010">
 +
===16 September===
2 µl of the mutagenesis product into 100 µl of competent cells
2 µl of the mutagenesis product into 100 µl of competent cells
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<div id=" .2F17_September_2010">'''17 September'''
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<div id=" .2F17_September_2010">
 +
===17 September===
- finalized project abstract
- finalized project abstract
-
<div id=" .2F19_September_2010">'''19 September'''
+
<div id=" .2F19_September_2010">
 +
===19 September===
- sent off project abstract to HQ
- sent off project abstract to HQ

Revision as of 01:11, 24 September 2010


Notebook

May
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
June
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
July
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
August
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31
September
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
October
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31


28 June

Did OH&S training and PC2 training

29 June

Began research on what we were going to use to promote taq

30 June

Looked at T7 with a Lac operon as a promotore of taq

13 July

Designed Primers for Taq into backbone

Researched about using an Asp/Pro between the peptide and the Taq

Began looking at ligation independent cloning for insertion of peptide

14 July

Looked at prices of Hotels in Boston

19 July

began looking at the structure of taq, its thermostability began looking at different mutations that can be done to taq to increase its thermostability without changing its structure

21 July

sent in sequence of wild type taq to iTasser

23 July

Checked thermostability of the mutations that will be done to Taq using Expasy

Sent in sequence of taq with mutations at 485, 515 and 540 to iTasser

3 August

Had iGEM team meeting

-Discussed ANZAAS Proposal

-Spoke about possibilities of using Geneart

-Discussed possible T-shirt designs

5 August

Meet up with Damian, discussed different strategies to use in modelling, began using YASARA to do our Modelling

10 August

Had iGEM team meeting

-Took Team Photo

===11 August===

PCR of Taq

Taq with a DNA concentration of 40ng/µL was diluted to 1µM by addition of 24µL of MilliQ water to 1µL of the Taq plasmid. Primers were diluted to 100µM by adding 354µL to the forward primer with a concentration of 35.4 nmol and 405µL to the reverser primer with a concentration of 40.5nmol. The primers were further diluted to 0.2µM by adding 10µL of the primer to 490µL of milliQ water. The PCR reaction was then made by adding 25µL of Master Mix, 2.5µL of each primer, 5µL of Rnase free water and 15µL of 0.2µM Taq Plasmid.

The PCR

98º 60 Sec 1 cycle

98º 15 Sec then 72º 90 Sec 25 cycle

72º 5 min 1 cycle

16 August

Collaborated with the Sheffield team by participating in an over the phone survery about Human Practices

17 August

had team meeting

- discussed how we can get more sponsors

- talked about placing our order to gene art

- began filling out the form to send Pennstates survey to the ethics committee

- discussed results for modelling

- decided to fix optimise our previous PCR

18 August

called few companies seeking sponsorship enquired for booking flights and accomadation

19 August

Working on comparing TAQ wild structure to the TAQ Mutated structure to see that there are no major differences, which will mean that TAQ is inactive but will retain it's thermostability at high temperatures.

Wrote an email to ABC TV, wanting to know if they will do piece on our project for there The New Inventors Program.

Waiting for results from I-Tasser on the mutated TAQ sequence. Went into the lab and made SOC media and LB- agar plates

20 August

Did two PCR reaction and a transformation The first PCR we ran was a touch down PCR. Temperatures ranged from 69 to 64. The second PCR was a two step PCR.

30 August

Ordered the TAQ polymerase from Mr. Gene. Should have it in 15 days. Meeting today at 10am to discuss project budget and costings. After the meeting we all are sitting together for a writing session.

31 August

Spent the morning organising our flights In the afternoon we all sat together for a writing session

1 September

We all sat together for another writing session.

7 September

Had a team meeting

14 September

Had a team meeting and made a lab time table, we also set out tasks which need to be done by the next meeting

  • Update the wiki
  • Finalise the form for the ethics committee for pennstate
  • Call up possible sponsors
  • Collect Tshirts
  • Work on abstract and team Roster
  • research heat liable bonds
  • order peptides
  • chat with emilio about possible bio assays
  • work on making the promotor part

15 September

Mutagenesis Quikchange XL kit

  • Template Concentration = 8ng/µl
  • Fwd 77ng/µl (low 260/280)
  • Rev 86.5 ng/µl (low 260/280)

We optimised for low 260/280

  • 5 µl buffer
  • 1.9 µl template
  • 1.7 µl Fwd
  • 1.62 µl Rev
  • 1 µl dNTP
  • 38.78 µl H2O --> reaction volume 50 µl
  • 1 µl Pfu

First mutagenesis was to turn the -35 site in the promoter region into an Afi II restriction site. Dpn1 digest (2 µl) for 2hrs

16 September

2 µl of the mutagenesis product into 100 µl of competent cells Plate 50 µl, 100 µl and 150 µl samples.


17 September

- finalized project abstract

19 September

- sent off project abstract to HQ
Retrieved from "http://2010.igem.org/Team:RMIT_Australia/Notebook"