September

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09-10-2008


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.

09-11-2008

11.09.08 Test Fura staining-Freigem08.jpg

11.09.08 Test Bindung Origami to alexa-Freigem08.jpg


09-12-2008

1) Origami with NIP and fluorophor for the binding measurement

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

  • 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

 

Origami with NIP (6x (1:5)) [µl]

Origami without NIP (6x (1:5)) [µl]

Oligos-Pool

43,68

43,68

remainders

2,4

2,4

MgAc

1

1

Phage DNA (448,4 mg/µl)

33.6

33.6

NIP-Oligo

1,68

---------------------

Pool oligo without fluorophor

0,72

0,72

Oligo without NIP

-------------------

1,68

3) Master cycler

The origamis were produced in the mastercycler as explained before.

 4) Purification of the DNA Origami

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

09-15-2008

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

09-16-2008

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

09-18-2008

Transformation (Sabine)
-transformation with the ligation product.

09-19-2008

Transformation (Sabine)
-no colonies on the plates.

09-20-2008

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

09-21-2008

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

Freiburg08 FT3.png