Commons
PCR
| Name: Sophie
| Date: 17.08.11
|
| repitition of PCR 25.08.11
(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
project name: new 3A assembly with amp-vector
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
Ligation
| Name: Sophie
| Date: 17.08.11
|
| Continue from Date: 17.08.11 Name: Sophie
Experiment 3A assembly digestion
|
| Project Name: new 3A assembly with amp-vector
|
Procedure
PCR tube:
total volume 20 μl
- add H2O (17 μl -X-Y-Z)
- add 2 μl Ligase Buffer 10x
- add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
- add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
- Add 1 μl T4-DNA Ligase
- Incubate 10-30 min at room temperature
- heat for 20 minutes at 80°C
- store at -20°C or directly proceed to transformation
|
| Name of part
| Ratio Insert:Vector
= 3:1 or 1:1
| Volume (μl)
|
| X insert 1
| LOV-Tap
| 1:1
| 2
|
| Y insert 2
| NOT-Gate
| 1:1
| 2
|
| Z vector
| psB1A3
| 1:1
| 2
|
| H2O
|
|
| 11
|
Documentation:
Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.
| Doing this cloning again because the Tet-vector makes problems.
Samples stored in Minipreps, verdaut-box and in Ligations-box
Name of the ligation-product: ♥-A3
|
Transformation
| Name: Sophie
| Date: 17.08.11
|
| Continue from Date: 17.08.11 Name: Sophie
Experiment: Ligation
|
| Project Name: new 3A assembly with Amp-vector, Blue light receptor
|
Procedure
- take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
- thaw cells on ice 20 minutes
- pipette 50 μl cells and 2 μl DNA into eppi still on ice!
- Incubate for 30 minutes on ice
- Heat at 42°C for 60 sec
- Incubate on ice for 5 minutes
- Add 200 μl LB Broth
- Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
- Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance
Documentation:
Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.
| Name: ♥-A3
stored in incubator on Amp-plates
vector: psBA3
inserts: LovTAP, NOT-Gate
|
red light receptor
transformation
Investigators:Julia
transformed e.coli with the ligation products from 16th of Aug.
It should be "Promotor+RBS (1/2)+ pcyA+ terminator"
and
"Promotor+RBS (1/2)+ ho1+ terminator"
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
"
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