Team:Freiburg/Notebook/18 August

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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

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.

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

Testdigest

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

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!

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

Investigators: Sophie

Digestion

Amount of DNA and H20:

Enzymes necessary for digestion:

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

Ligation

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

Documentation:

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

PCR

Investigators: Rüdiger

PCR did not work. New primer design.