Team:Freiburg/Notebook/9 September

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Revision as of 12:19, 10 September 2011 by SophieCramer (Talk | contribs)


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

Contents

Commons

Testdigests

Investigators: Sophie

Testdigest of our various minipreps

100Testdigests Freiburg.jpg


green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME


blue light receptor

PCR


Name: Sophie


Date: 9.9.11
Continue from Experiment: Gibson (Date): 9.9.11

(Name): Sophie, Sandra

Project Name:

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20 Name
10µl 5x Phusion Buffer of Primer
2.5µl Primer fw P97
2.5µl Primer dw P100
1µl dNTPs of Template DNA
1µl DNA-Template G-♥-NOT and G-♥-NOT 50
0.5 µl Phusion (add in the end)

What program do you use?

First 25 cycles: touchdown 69°C -0.4°C

next 5 cycles: touchdown 72°C -0.2°C


To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.


How did you label the PCR-Product, where is it stored and what do you do next?

Labeled: G-♥-NOT inserts a,b

stored in blue light box

Digestion


Name: Sophie Date: 9.9.11
Continue from Date 9.9.11 Name: Sophie

Experiment: PCR

Project Name: ♥-NOT in PR-vector

Procedure


  1. add H2O (38μl-DNA )
  2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
  3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
  4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
  5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
  6. heat for 1-2 hours 37°C (6 hours if time)
  7. heat for 20 minutes 80°C (inactivation of enzymes)
  8. keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:


Sample Name DNA concentration (μg/μl)
G-♥-NOT a,b ~140

Restriction enzymes you need to cut the vector, insert1 and insert 2:


Components
Vector (μl)
 Insert(μl)
DNA (500ng)
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl) Spe I Nhe I
Enzyme 2 (1μl) Pst I Pst I
H2O (38 μl- DNA)
In total 50 μl

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.


The LOV-Repressor protein needs to be expressible

Samples stored in blue light box

Vector used: all PR-vectors (D39- D44)

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME

                       

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