Team:Freiburg/Notebook/25 July

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Commons

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

1. 2Min 94°C
2. 30s 94°C
3. 30s 55°C
4. 3min 72°C
5. 10min 72°C

step 2,3 and 4 in 35 cycles

Labeled PSB 1 A3, PSB 1 C3, PSB 1 K3 and PSB 1 T3 stored in -20°C in last drawer

green light receptor

Testdigest: Ligation of CcaS into pSB1K3

Investigators:JULIA

Testdigest

For one reaction you need For Mastermix: 35 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.

Picture of 1% gel:

result:4a2,4b2,4b3,4b4,4b5 should have the right insert. Send 4a2 and 4b2 to GATC for sequencing.

blue light receptor

Gibson Not-Gate:Transformation

Investigators: Sophie

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.

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

"57°C auf 70°C" (first annealing temperature:55°c, after 10 cycles 65°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?

I labelled it with a heart and a star and stored it in the minipreps 3 box.

I will do Gibson assemblz of the two parts next.

DNA-concentration measured with nanodrop:

We will repeat the PCr, because of the bad result of S35. We will do a PCR with higher temperature.

Precipitator

Digestion

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:

Documentation:

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

Run a gel to verify if the part is cut out.