Team:Freiburg/Notebook/1 August

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green light receptor

Qickchange PCR of CcaS

Investigators:Julia
repeating experiment from___, because we had no positive transformation, probably chosen wrong annealing temperature before.

blue light receptor

PCR

Investigators: Sophie

As our last PCRs of the LovTAP with the Gibson overhangs didn't work well, we now try a PCR with primers without overhangs for Gibson assembly.

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

LovTAP ohne Ueberhaenge

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?

Labelled: M35 short; stored in Gibson Stuff box.

red light receptor

3A-assembly with pcyA and the terminator BBa_1006

Investigators:Julia

1.Digestion:

terminator (S21a), 26µl of the miniprep+ 12,1 µl H2O, cut with XbaI&PstI
pcyA (part BBa_I15009), 3 µl of miniprep + 35µl H2O, cut with EcoRI&SpeI
Vector-backbone (10 µl) pSB1T3, cut with EcoRI,PstI & DpnI

to each reation 5µl BSA(10x) and 5µl NEB buffer4 were added, digested at 37°C for two hours, inactivated at 80°C for 20min.

2.Ligation

2µl of digested pcyA,terminator and vector were added to 11µl H2O,2µl ligase buffer and 1µl T4 ligase.
Incubated at room temperature for 35 min, inactivated at 80°C for 20 min.

3.Transformation

Lysis cassette

Phage Lysis Cassette (K124017) + RBS (B0034)

Investigators: Theo

Digestion of 3A Assembly

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

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

Documentation:

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

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 etc.

Transformation

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.

Precipitator

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

Ruediger PCR (modified by Tobi and me. First 10 cycles with 55°C, next cycles with 62°C annealing temperature.

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?

3A-assembly

1.Digestion

Investigators: Theo

Digestion of PCR product.

• S39
• S43

were digested with EcoRI and SpeI

• P18
• P19
• P20

were digested with XbaI and PstI.

2.Ligation

Investigators: Theo

Ligation of:

• S39+P18
• S43+P18
• S39+P19
• S43+P19
• S39+P20
• S43+P20

stored in Theo´s box.