Commons
PCR
| Name: Sophie
| Date: 17.08.11
|
| Continue from Experiment (Date)
(Name): Commons
|
| Project Name: tet-vector seems to make problems -> new PCR with original psB1T3-DNA
|
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl
| H20
| Name
|
| 10µl
| 5x Phusion Buffer
| of Primer
|
| 2.5µl
| Primer fw
| SB-prep-3P-1
|
| 2.5µl
| Primer dw
| SB-prep-2Ea
|
| 1µl
| dNTPs
| of Template DNA
|
| 1µl
| DNA-Template
|
PSB 1 T3
|
| 0.5 µl
| Phusion (add in the end)
|
|
What program do you use?
- 2Min 94°C
- 30s 94°C
- 30s 55°C
- 3min 72°C
- 10min 72°C
step 2,3 and 4 in 35 cycles
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 PSB 1 T3 stored in -20°C in last drawer
green light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
blue light receptor
Miniprep
Investigators: Sandra
Miniprep of LovTAP-NotGate-T3:
stored in undigested miniprep box II
Testdigest
Investigators: Sandra
Testdigest of Miniprep product.
Enzymes:
3A-assembly of Not-Gate, LovTAP in pSB1A3
Investigators: Sophie
As the Tet-vector seemed to make problems, we did a 3A assembly again, this time with amp-vector.
Digestion
Amount of DNA and H20:
| Sample
| DNA μl
| H20 μl
|
| LovTAP
| 5
| 33
|
| Not-Gate
| 2
| 36
|
| pSB1A3
| 20
| 18
|
Enzymes necessary for digestion:
|
| LovTAP
| Not-Gate
| vector
|
| enzyme 1
| EcoRI
| XbaI
| EcoRI
|
| enzyme 2
| NheI
| PstI
| PstI
|
- Incubation at 37°C for 6 hours
- 20 minutes at 80°C
red light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
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