Team:Freiburg/Notebook/22 July

From 2011.igem.org

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Procedure:
 
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1.Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm
 
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(6800 x g) for 3 min at room temperature (15–25°C).
 
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2.Resuspend pelleted bacterial cells in 250 ␣l Buffer P1 and transfer to a microcentrifuge tube.
 
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3.Add 250 ␣l Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
 
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4.Add 350 ␣l Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
 
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5.Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
 
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6.Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting.␣Centrifuge for 30–60 s and discard the flow-through, or␣apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source.
 
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7.Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB. ␣Centrifuge for 30–60 s and discard the flow-through, or␣apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source.
 
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Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
 
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8.Wash the QIAprep spin column by adding 0.75 ml Buffer PE. ␣Centrifuge for 30–60 s and discard the flow-through, or␣apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source. Transfer the QIAprep spin column to the collection tube.
 
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9.Centrifuge for 1 min to remove residual wash buffer.
 
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10.Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 ␣l Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
 
'''Documentation:'''
'''Documentation:'''

Revision as of 16:33, 1 August 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

Contents

Precipitator

Miniprep

Qiagen Kit


Name:

Sophie

Date:

22.07

Continue from Experiment (Date) Trafo 20.07 Ruediger

(Name)

Project Name:

GFPpbd

Documentation:

Why are you doing this experiment? Name the parts which you extract.


1abc: S39+M14+P28+P18

2abc: S39+M14+P28+P19

3abc: S39+M14+P28+P20

4abc: S43+M14+P28+P18

5abc: S43+M14+P28+P19

6abc: S43+M14+P28+P20


all CM resistance ps131C3



Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results.


Mistakes: some lids broke

nanodrop labeling partly confused

Sample ID Nucleic Acid Conc. Unit 260/280 260/230
1a
60.6
 ng/µl
1.85
1.75
1b
102.8
 ng/µl
1.66
0.96
1c
41.5
 ng/µl
1.74
1.63
1d
85.5
 ng/µl
1.58
1.09
2a
72.4
 ng/µl
1.62
1.04
2b
9.7
 ng/µl
0.86
0.34
2c
116.8
 ng/µl
1.64
0.9
2d
38.5
 ng/µl
1.56
0.95
3a
86.5
 ng/µl
1.56
0.75
3b
77.9
 ng/µl
1.57
0.89
3c
94
 ng/µl
1.66
1.07
3d
88.1
 ng/µl
1.64
0.95
4a
91.3
 ng/µl
1.64
0.91
4b
93.4
 ng/µl
1.67
1.09
4c
111.5
 ng/µl
1.7
1.15
4d
71.5
 ng/µl
1.86
1.95
5a
73.2
 ng/µl
1.81
1.75
5b
172.3
 ng/µl
1.79
2.01
5c
53
 ng/µl
1.61
1.98
5d
97
 ng/µl
1.72
1.43
6a
92
 ng/µl
1.7
1.27
6b
92.8
 ng/µl
1.62
1.05
6c
81.3
 ng/µl
1.66
1.14
6d
106.8
 ng/µl
1.73
1.26


Testdigest


Name:

Ruediger

Date:

21.07

Continue from Experiment (Date)

Miniprep Sophie (Name)

Project Name: GFPPbd

For one reaction you need: For Mastermix: Number of samples+2extra


4μl H2O 104
1μl Buffer, NEB4 26
1μl BSA (10x) 26
0,5 μl Enzym 1
0,5 μl Enzym 2
3 μl DNA

DNA from todays Miniprep. See labeling there.


10 μl total volume


Give 3 μl of DNA in an eppi and add 7μl of the mastermix.

Incubate for about 1h at 37°C.


Add 1 μl Loading dye buffer and load the gel.

Take a picture of the gel, print picture and label the lanes!


Freiburg11 7 23 2011 8 07 30 PM.Jpg

Meeting

attendants: Jakob, Julia, Manuel, Ruediger, Sandra, Sophie, Theo (later), Tobi time: 9:00 - 10:30

green light receptor

already done:

  • CcaS was amplified again from the genomic DNA

To-do:

  • 3-A-Assembly of CcaR into an empty vector with a medium promotor
  • 3-A-Assembly of CcaS into an empty vector with a weak promotor
  • 3-A-Assembly of CcaR and CcaS to get the final construct

blue light receptor

already done:

  • Primer-Design for the Gibson-Assembly
  • growth medium without tryptophane is already ordered

To-do:

  • amplify the NOT-Gate

red light receptor

already done:

  • one positive colony from the 3-A of ho1 and the terminator
  • no positive colony from the 3-A of pcyA and the terminator
  • the PCR to amplify cph8 didn't give a product, we asked the team from Mexico to send us the cph8
  • the team from Upsala (Sweden) has succeeded in amplifying the cph8 (according to their notebook)

To-do:

  • 3-A-Assembly of the ho1-term with a mediumPromotor-mediumRBS


Lysis cassette

already done:

  • the quick change didn't work the first time, we should repeat this
  • possible alternative to express the lysis cassette could be: temperature regulated RNA

To-do:

  • repeat the quick change

Precipitator

already done:

  • the GFP-PBD was cloned (colonies showed up)


To-do:

  • test digest and sequencing of the GFP-PBD (first have a look if you see the GFP fluorescence)
  • possible test of the PBD: grow the E. coli, lyse them, put the lysate into a plate (96 well?), wash the wells to get rid of the other proteins, measure the GFP fluorescence with a plate reader
  • microscope the E. coli cells

other suff

  • think about possible give-aways for the sponsoring video
  • jacob will create a Youtube account and upload the sponsoring video
  • meeting for the wiki takeover: wednesday 9:00 am (new time: 12:15)
  • modelling should be started now, either Ni-binding and release or GFP-PBD binding and release, Ruediger will do this



                       

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