Team:Freiburg/Notebook/8 September

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green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

Troubleshooting of the modified Lysis genes K124017

Investigators:Theo

Transformation of M66 with M48 did not work. Probably because the stock of M66 that we made was indeed another part.
So S4+S15 Nr1 is going to be Nr1 and S4+S15 Nr7 is Nr7.
M48+1 and M48+2 as well as just Nr1 and Nr2 were inoculated so that they could be sent for sequencing on Friday

Precipitator

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.

Miniprep

Qiagen Kit

Procedure:

The following procedures are carried out at a room temperature. All centrifugation steps should be performed between 11,000 – 16,000 x g

1.Centrifuge 0.5 – 5 ml of bacterial culture in a clear 1.5 ml tube at full speed for 15 – 20 seconds in a microcentrifuge. Discard supernatant.

2.Add 200 µl of P1 Buffer (Red) to the tube and resuspend pellet completely (i.e., by vortexing or pipetting).

3.Add 200 µl of P2 Buffer (Green) and mix by inverting the tube 2 – 4 times. Cells are completely lysed when the solution appears clear, purple and viscous. Proceed to the next step within 1-2 minutes.

4.Add 400 µl of P3 Buffer (Yellow) and mix gently but thoroughly. Do not vortex. The sample will turn yellow when the neutralization is complete. Allow the lysate to incubate at room temperature for 1-2 minutes.

5. Centrifuge sample(s) for 2 minutes.

6.Place a Zymo-Spin IIN column in a Collection Tube and transfer the supernatant from step 5into the Zymo-Spin IIN column. When pipetting the supernatant be careful not to disturb the green pellet to avoid transferring anz cellular debris to the column.

7.Centrifuge the Zymo-Spin IIN/Collection Tube assembly for 30 seconds.

8.Discard the flow-through in the Collection Tube, making sure the flow-through does not touch the bottom of the column. Return the Zymo-Spin IIN column to the Collection Tube

9.Add 200 µl of Endo-Wash-Buffer to the column and centrifuge for 30 seconds.

10. Add 400 µl of Plasmid Wash Buffer to the column. Centrifuge for 1 minute.

11. Transfer the column into a clean 1.5 ml microcentrifuge tube and then add 30 µl of DNA Elution Buffer to the column. Centrifuge for 30 seconds to elute the plasmid DNA.

Documentation:

Why are you doing this experiment? Name the parts which you extract.

Prepped all colonies from yesterday's transformation.

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.

NAME OF YOUR EXPERIMENT

Investigators: NAME