September

From 2008.igem.org

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Revision as of 18:54, 9 October 2008

__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 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

@@ Line 8: / Line 8: @@
 <h3>09-10-2008</h3>
 <br>
-'''CMV PCR'''<br>
+'''CMV PCR''' (Sabine)<br>
-I did a PCR with primer for the CMV promotor. As DNA template I used the CMV+RLuc construct from the Ljubljana group which I isolated from the parts collection 2007.<br>
+-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.<br>
-For a 50 µl reaction I used:<br>
+For a 50 µl reaction:<br>
 ,4 µl H2O<br>
 µl buffer (10x)<br>
@@ Line 29: / Line 29: @@
 . HOLD  6°C<br>
-I got no product.
+No product was received.
 <h3>09-11-2008</h3>
@@ Line 294: / Line 294: @@
 </html>
-'''5) Digestion of CMV+Rluc'''<br>
+'''5) Digestion of CMV+Rluc''' (Sabine)<br>
-Digestion with EcoRV und FspI(3h at 37°C)
+Digestion with EcoRV und FspI (3h at 37°C)
 <ul>
 <li> 5µl plasmid
@@ Line 305: / Line 305: @@
 [[image:Verdau_CMVRluc_EcoRV_FspI_klein.jpg]]<br>
-'''6) CMV PCR'''<br>
+'''6) CMV PCR''' (Sabine)<br>
-I tried the same PCR with different annealing temperatures. The optimal annealing temperature is 62°C. I used a gradient between 58°C and 62°C. I again got no products.
+-the same PCR with different annealing temperatures using a gradient between 58°C and 62°C (optimal annealing temperature: 62°C)<br>
+-again, no products were gained
 <h3>09-15-2008</h3>
-'''CMV PCR'''<br>
+'''CMV-PCR''' (Sabine)<br>
-I again tried the CMV PCR, but this time with different polymerases. I used the Taq Polymerase and a Mix. I also tried one approach with the complete plasmid and one with the digested plasmid (see digestion of CMV+Rluc)
+-another effort to gain the CMV-Promotor via PCR, this time with different polymerases: Taq Polymerase and a Mix.<br>
-I got products in the approach with taq polymerase as well as in the approach with the Mix. I cut out 3 bands from each approach.
+-one approach with the complete plasmid and one with the digested plasmid (see digestion of CMV+Rluc)<br>
+-products in the approach with taq polymerase as well as in the approach with the Mix. 3 bands from each approach were cut out
 <h3>09-16-2008</h3>
-'''Gel purification'''<br>
+'''Gel purification''' (Sabine)<br>
-I did a gel purification of the PCR products.
+-gel purification of the PCR products
-'''Digestion of the PCR products and the transfectionvector'''<br>
+'''Digestion of the PCR products and the transfectionvector''' (Sabine, Kathrin)<br>
-Digestion with EcoRI and PstI. Then Kathrin did a ligation.
+-digestion with EcoRI and PstI  <br>
+-ligation
 <h3>09-18-2008</h3>
-'''Transformation'''<br>
+'''Transformation''' (Sabine)<br>
-I did a transformation with the ligation product.
+-transformation with the ligation product.
 <h3>09-19-2008</h3>
-'''Transformation'''<br>
+'''Transformation''' (Sabine)<br>
-There were no colonies on the plates.
+-no colonies on the plates.
 <h3>09-20-2008</h3>
-'''Digestion of the PCR products and the transfectionvector'''<br>
+'''Digestion of the PCR products and the transfection-vector''' (Sabine)<br>
-Now I used the restrictionenzymes XbaI and SpeI.
+-the restriction-enzymes XbaI and SpeI were used
-'''Gel purification and ligation'''<br>
+'''Gel purification and ligation''' (Sabine)<br>
-The digested PCR products and the vector.
+-digestion of PCR products and vector
-<h3>09-21-2008</h3>
+<h3>09-21-2008 </h3>
-'''Transformation of the ligation'''<br>
+'''Transformation of the ligation''' (Sabine)<br>
-I used 10 µl of the ligation and used RV 308 cells
+-RV 308 cells were transformed with 10 µl of the ligation
 }}