Team:Freiburg/Notebook/9 September

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Commons

Testdigests

Investigators: Sophie

Testdigest of our various minipreps

green light receptor

digest for cloning PcpcG infront of CFP/YFP

Investigators:Julia

1. Digestion of PCR Product(PcpcG)

38µl PCR product
5µl BSA (10x)
5µl NEB buffer 4
1µl EcoRI
1µl SpeI

2.Digestion of CFP/YFP Vector

36,1/34 µl water
1.9µl DNA of YFP/ 3.7µl DNA of CFP = 500ng DNA
5µl BSA (10x)
5µl NEB buffer 4
1µl EcoRI
1µl XbaI

Incubation over night.

blue light receptor

Gibson assembly

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.

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

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

First 25 cycles: touchdown 69°C -0.4°C

next 5 cycles: touchdown 72°C -0.2°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?

Labeled: G-♥-NOT inserts a,b

stored in blue light box

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:

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.

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME