Team:Freiburg/Notebook/31 August

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

• < home >
• < team >
  • → members
  • → attributions
  • → freiburg
• < project >
  • → description
  • → results
  • → modeling
  • → notebook
  • → sample data
• < human practices >
  • → oath
  • → ethics
  • → safety
• < gallery >
• < sponsors >
• < to the forums >

Contents 1 green light receptor 1.1 NAME OF YOUR EXPERIMENT 2 blue light receptor 2.1 Miniprep 2.2 Testdigest 3 red light receptor 3.1 NAME OF YOUR EXPERIMENT 4 Lysis cassette 4.1 NAME OF YOUR EXPERIMENT 5 Precipitator 5.1 Ligation 5.2 Transformation 5.3 PCR 5.4 Digestion

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

Miniprep

Investigators: Sandra

Miniprep of:

• ♥-A3 Not 1
• ♥-A3 noT 2
• ♥-A3 noT 3

On the plates with tetracyclin nothing grew.

Testdigest

Investigators: Sandra

Testdigest of minipreps.

Digested with EcoRI and PstI.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

Ligation

Name: Sophie	Date: 31.01.11
Continue from Date: 30.08.11 Name: Sophie Experiment: Digestion
Project Name: inducible promoter for pbd

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
Y insert 2	ε 5
Z vector	psb1C3
H2O

Documentation:

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

See Digestion...

Transformation

Name:Sophie	Date: 31.08.11
Continue from Date: 31.08.11 Name: Sophie Experiment: Ligation
Project Name: inducible promoter for pbd

Procedure

1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
2. thaw cells on ice 20 minutes
3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
4. Incubate for 30 minutes on ice
5. Heat at 42°C for 60 sec
6. Incubate on ice for 5 minutes
7. Add 200 μl LB Broth
8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
9. Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

Documentation:

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.

Name: S54-ε 5-C3 1:1 / 1:3 stored in incubator on Cm plates

PCR

Name: Sophie	Date: 31.08.11
Continue from Experiment new (Date) (Name)
Project Name:: pbd in Gst-vector

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	P93
2.5µl	Primer dw	P94
1µl	dNTPs	of Template DNA
1µl	DNA-Template
0.5 µl	Phusion (add in the end)

What program do you use?

First 25 cycles touchdown 63°C -0,3°C per cycle

next 10 cycles touchdown 72°C -0,3°C per cycle

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 GFP-pbd-PCR

stored in

I will digest it with Dpn I and with bamH I and Xho and ligate it to a GST-vector.

Digestion

Name: Sophie	Date: 31.08.11
Continue from Date: 31.08.11 Name: Sophie Experiment: PCR
Project Name: pbd into GST-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. add 5,6 μl phosphatase buffer and 1 μl antarctic phosphatse to the vector and digest for another hour
8. heat for 20 minutes 80°C (inactivation of enzymes)
9. 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)
pGex	480
gFP-pbd-PCR

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

Components	Vector (μl)		Insert
DNA (500ng)
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl)		BamH I		BamH I
Enzyme 2 (1μl)		Xho I		Xho 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.

Why? I want to produce pbd-protein to make experiments and to get data for the modeling. Vector: pGEX from Hemin insert: GFP-pbd-PCR