Team:Freiburg/Notebook/17 August

• < home >
• < team >
  • → members
  • → attributions
  • → freiburg
• < project >
  • → description
  • → results
  • → modeling
  • → notebook
  • → sample data
• < human practices >
  • → oath
  • → ethics
  • → safety
• < gallery >
• < sponsors >
• < to the forums >

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

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?

Labeled PSB 1 T3 stored in -20°C in last drawer

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

Miniprep

Investigators: Sandra

Miniprep of LovTAP-NotGate-T3:

• 15ng/μl

stored in undigested miniprep box II

Testdigest

Investigators: Sandra

Testdigest of Miniprep product.

Enzymes:

• EcoRI
• PstI

3A-assembly of Not-Gate, LovTAP in pSB1A3

Investigators: Sophie
project name: new 3A assembly with amp-vector

As the Tet-vector seemed to make problems, we did a 3A assembly again, this time with amp-vector.
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 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.

red light receptor

transformation

Investigators:Julia
transformed e.coli with the ligation products from 16th of Aug.
It should be "Promotor+RBS (1/2)+ pcyA+ terminator"
and
"Promotor+RBS (1/2)+ ho1+ terminator"

Investigators: Jakob

• PCR from yesterday was negative

• New PCR (BBa_I15010) (protocol see 16.08.2011)

• Transformation (BBa_I15010)

Lysis cassette

2A Assembly

Investigators:Theo

Colonies picked

Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME