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__september
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
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 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
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Origami with NIP (6x (1:5)) [µl]
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Origami without NIP (6x (1:5)) [µl]
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Oligos-Pool
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43,68
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43,68
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remainders
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2,4
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2,4
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MgAc
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1
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1
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Phage DNA (448,4 mg/µl)
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33.6
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33.6
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NIP-Oligo
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1,68
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---------------------
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Pool oligo without fluorophor
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0,72
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0,72
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Oligo without NIP
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-------------------
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1,68
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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
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