Team:Freiburg/Notebook/18 August

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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 18 August </a>
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==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
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==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===2A assembly===
-
'''Investigators:NAME'''
+
'''Investigators:Theo'''
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Sent for sequencing
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==

Latest revision as of 01:09, 22 September 2011


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


Contents

green light receptor

Quickchange

Investigators:Jakob

blue light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



red light receptor

picked colonies from yesterdays trnasformation.

new PCR in order to again cph8 from pJT122.
last time PCR did not work.

Lysis cassette

2A assembly

Investigators:Theo


Sent for sequencing

Precipitator

Miniprep

zymo Kit


Name:Sophie


Date:18.08.11
Continue from Experiment: Cloning: ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too. (Date): 12.08.11

(Name): Sophie

Project Name: inducible Promoter with pbd with cm-vector


Documentation:

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

ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too.

Name of the parts: GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2


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


GFP-pbd 4 4 4: 164,1 ng/µl

GFP-pbd 4 1 3: 100,8 ng/µl

GFP-pbd 6 6 8: 163,5 ng/µl

GFP-pbd 4 2 2: 144,4 ng/µl

How did you label your probes and where are they stored?


Labelled GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2

stored in -20 until sequencing


Testdigest

Name: Sophie


Date: 18.08.11
Continue from Experiment: Miniprep (Date): 18.08.11

(Name): Sophie

Project Name:inducible promoter for pbd with cm-vector

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


4μl H2O 20
1μl Buffer, NEB4 5
1μl BSA (10x) 5
0,5 μl Enzym 1 2,5
0,5 μl Enzym 2 2,5
3 μl DNA

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!
Freigem18.8.11.jpg

3A-assembly of IPTG-promoter, pbd-GFP and Cm-vector


Investigators: Sophie

Digestion

Amount of DNA and H20:

Sample DNA μl H20 μl
IPTG-Promoter 2,5 33
GFP-pbd 3 36
pSB1A3 20 18


Enzymes necessary for digestion:

GFP-pbd: 4 4 4 or 6 6 8 IPTG-Promoter: S54 vector: psB1C3
enzyme 1 EcoRI XbaI EcoRI
enzyme 2 NheI PstI PstI


  • Incubation at 37°C for 6 hours
  • 20 minutes at 80°C



Ligation

Name: Sophie Date: 18.08.11
Continue from Date: 18.08.11 Name: Sophie

Experiment: Cloning

Project Name: inducible Promoter for pbd with Cm-vector

Procedure


PCR tube:

total volume 20 μl


  1. add H2O (17 μl -X-Y-Z)
  2. add 2 μl Ligase Buffer 10x
  3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
  4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
  5. Add 1 μl T4-DNA Ligase
  6. Incubate 10-30 min at room temperature
  7. heat for 20 minutes at 80°C
  8. store at -20°C or directly proceed to transformation


Name of part Ratio Insert:Vector

= 3:1 or 1:1

Volume (μl)
X insert 1 S54 both 2 or 3
Y insert 2 PR 444 bzw. PR 668 2 or 3
Z vector Psb1A3 2 or 1
H2O 11

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation


Stored in Ligation-box, single parts stored in minipreps, verdaut-box

name of parts see above.

PCR

Investigators: Rüdiger

name templates primer
a pGex-6-P-1a P62+P63
b pGex-6-P-1b ´´
a10 pGex-G-P-1a ´´
b10 pGex-6-P-1b ´´

PCR did not work. New primer design.

                       

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