Team:Freiburg/Notebook/14 July

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red light receptor

3A-assembly: Digest

Investigators: Jakob

3A-assembly of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator)

Digestion

Procedure

1. add H2O (16μl-DNA )   

2. 2μl NEB4 buffer

3. 2 μl 10x BSA     (used 1:10 diluted )

4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)

5. 1 μl restriction enzymes

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:

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

3A-assembly: Ligation and transformation

Investigators: Julia

Ligation of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator) in vector pSB1T3.

L23: Ligation of S23+S21a in pSB1T3 L22: Ligation of S22+S21a in pSB1T3 Ligation

Procedure

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

Competent neb10 strain was transformed with 2μl ligation reaction and plated out.

PCR

Investigators: Julia

PCR of the red light receptor, cph8.

Primer:

DNA template:

• BBa_I150010

Programm: 2min 55/60

Lysis cassette

Quickchange PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

Precipitator

Test-digest

Investigators: Rüdiger

Testdigest of the minipreps, Sophie did (june, 13).

• Enzyme 1: PstI
• Enzyme 2: EcoRI

Image of different samples from miniprep.