September

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Sep. 10th 2008

CMV PCR (Sabine)
-PCR with primer for the CMV promotor; as DNA template the CMV+RLuc construct from the Ljubljana group (isolated from the parts collection 2007) was used.

For a 50 µl reaction:
40,4 µl H2O
5 µl buffer (10x)
1,5 µl FWD Primer (15pmol)(
1,5 µl REV Primer (15pmol)
1 µl dNTP (10mM)
0,1 µl DNA template
0,5 µl Pfu Polymerase

The settings for the PCR machine are the following:
1. T=94°C 00:02:00
2. T=94°C 00:00:30
3. T=62°C 00:00:30
4. T=72°C 00:01:00
5. GOTO 2 REP 29
6. T=72°C 00:10:00
7. HOLD 6°C

No product was received.

Sep. 11th 2008

Sep. 12th 2008

1) Origami with NIP and fluorophor for the binding measurement (Norman+Simone)

We had to produce some new origami for our next binding measurements.

• Origami with NIP and fluorophor

• Origami only with fluorophor (without NIP); negative control

see at the protocol from 07-24-2008

2) Origami for the Calciummeasurement (Norman+Simone)

• Origami with NIP

• Origami without NIP (negative control)

see at the protocol from 07-24-2008

To increase the concentration of origami we also made to probes with the double amount

ingredients of the protocol from 07.24.2008

3) Master cycler (Norman+Simone)

The origamis were produced in the mastercycler as explained before.

4) Purification of the DNA Origami (Norman+Simone)

Was done as before

5) Digestion of CMV+Rluc (Sabine) Digestion with EcoRV und FspI (3h at 37°C)

• 5µl plasmid
• 10µl H2O
• 0,5µl enzyme
• 2,5µl buffer (2)
• 0,5µl BSA

File:Verdau CMVRluc EcoRV FspI klein.jpg

6) CMV PCR (Sabine)
-the same PCR with different annealing temperatures using a gradient between 58°C and 62°C (optimal annealing temperature: 62°C)
-again, no products were gained

Sep. 15th 2008

CMV-PCR (Sabine)
-another effort to gain the CMV-Promotor via PCR, this time with different polymerases: Taq Polymerase and a Mix.
-one approach with the complete plasmid and one with the digested plasmid (see digestion of CMV+Rluc)
-products in the approach with taq polymerase as well as in the approach with the Mix. 3 bands from each approach were cut out

Sep. 16th 2008

Gel purification (Sabine)
-gel purification of the PCR products

Digestion of the PCR products and the transfectionvector (Sabine, Kathrin)
-digestion with EcoRI and PstI
-ligation

Sep. 18th 2008

Transformation (Sabine)
-transformation with the ligation product.

Sep. 19th 2008

Transformation (Sabine)
-no colonies on the plates.

Sep. 20th 2008

Digestion of the PCR products and the transfection-vector (Sabine)
-the restriction-enzymes XbaI and SpeI were used

Gel purification and ligation (Sabine)
-digestion of PCR products and vector

Sep. 21st 2008

Transformation of the ligation (Sabine)
-RV 308 cells were transformed with 10 µl of the ligation

Sep. 23rd 2008

1. Transformation of the 4 plasmids of ATG (michael)
-> Signalpeptide
-> Transmembrane domain
-> His-tag
-> Strep-tag

2. Digestion...

Sep. 24th 2008

1. Transformation (michael)
of the Ligations: CMV-Promotor + Vector
proportion: PCR-product (CMV promoter) / vector
-> 6/2
-> 4/4

2. Picking clons of the '4 ATG trafos' (michael)
-> Signalpeptide
-> Transmembrane domain
-> His-tag
-> Strep-tag
after growing glycerinstocks and Miniprep of all 4

3. Picking clons from stock (michael) -> lipocalin
-> C-GFP and N-GFP
-> C-CFP and N-CFP
-> scFv anti NIP

4. Digestion of the CMV-promoter
Approach Mix: 3/4.1/4.2/4.3/4.4

...

Sep. 25th 2008

Miniprep (normann)
of C-CFP and scFv-anti-NIP

Picking from stocks (normann):
N-CFP, N-GFP, C-CFP, C-GFP

Defrost B12-cells (michael)
and put in new RPMI:
->500ml RPMI
+50ml FCS(10%)
+5ml Hepes(1M)
+5ml L-glutamine(200mM)
+5ml Pen-Strep
+1,75µl ß-Mercaptoethanol(14.3M)

Ligation 2nd try (sabine)
with T4-ligase:
Insert: CMV-Promotor, old and new PCR-Product
Vector: Transfection-Vector fermented with EcoRI/SpeI
-Approaches (for each, old and new PCR-Product): Insert/Vector -> a) 6/2, b) 4/4

Sep. 26th 2008

Transformation of the 'ligation 2nd try' (michael)

Picking clones
4x Signalpeptide
2x Transmembraneregion

Miniprep of: (norman)
-Lipocalin
-Venus split N-GFP
-Venus split C-GFP
-Cerulan split N-CFP