Team:Freiburg/Notebook/31 August

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

green light receptor

Digestion for 2A-assembly

Investigators: Julia, Sandra

Digestion of:

total volume was 50 µl , 1 µl Alcalic Phosphotase and its buffer
was added after 3 hours of incubation to the vector-plasmids S2 and S4

Ligation

Ligation of the parts:

in vector S2b, which is part BBa_J23110(strong promotor):

• S64 (Ccas)
• S67 (Ccar)

in vector S4,which is part BBa_B0034(RBS):

• S46 (ho1)
• S50 (pcyA)

It was incubated over night at 16°C. In the morning ligase was inactivated at 80°C.

Transformation on 1.09.2011

for results see 3.09.2011

blue light receptor

Miniprep

Investigators: Sandra

Miniprep of:

• ♥-A3 Not 1
• ♥-A3 noT 2
• ♥-A3 noT 3

Plates with tetracyclin showed not a single colony.

Testdigest

Investigators: Sandra

Testdigest of minipreps.

Digested with EcoRI and PstI.

Testdigestd showed no bands for the inserts.

new Ligation

Investigators: Sandra

New Ligation with the already digested parts. New digested vector from Julia, pSB1C3.

Parts were ligated for 2 hours this time.

Parts:

• Notgate
• Lovtap
• vector: pSB1C3

Trafo

Investigators: Sandra

After the ligation a transformation was performed and the plates were incubated over night at 37°C. Hopefully we get something to see tomorrow:-)

Plan B: We designed new primers for the gibson assembly.

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

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?

EcoR1+suffix/not

Prefix+suffix/not

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

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?

Gibson did not work because DNA concentration was too high.

Gibson-Assembly

1. Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:

3 ml of 1 M Tris-HCl pH 7.5150 μl of 2 M MgCl₂60 μl of 100 mM dGTP 60 μl of 100 mM dATP 60 μl of 100 mM dTTP60 μl of 100 mM dCTP300 μl of 1 M DTT 1.5 g PEG-8000300 μl of 100 mM NADAdd water to 6 ml Aliquot 100 μl and store at -20 °C

2. Prepare an assembly master mixture. This can be prepared by combining the following:

320 μl 5X ISO buffer0.64 μl of 10 U/ μl T5 exo20 μl of 2 U/μl Phusion pol160 μl of 40 U/μl Taq ligAdd water to 1.2 ml

Aliquot 15 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles. This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.

3. Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.

4. Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).

5. Incubate at 50 °C for 15 to 60 min (60 min is optimal).

6. If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.

Documentation:

Why are you doing this experiment? Name the parts for the Gibson-Assembly.

Probes

Describe your results and mistakes. Did you digest it? Results?

How did you label your samples and where are they stored?

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:

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

Documentation:

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.

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

First 25 cycles touchdown 63°C -0,3°C per cycle

next 10 cycles touchdown 72°C -0,3°C per cycle

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 GFP-pbd-PCR

stored in

I will digest it with Dpn I and with bamH I and Xho and ligate it to a GST-vector.

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. add 5,6 μl phosphatase buffer and 1 μl antarctic phosphatse to the vector and digest for another hour
8. heat for 20 minutes 80°C (inactivation of enzymes)
9. 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:

Documentation:

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