Team:Freiburg/Notebook/31 August

From 2011.igem.org

(Difference between revisions)
(→Precipitator)
(→Precipitator)
Line 164: Line 164:
|}
|}
 +
'''PCR'''
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Sophie
 +
 +
 +
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 31.08.11
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment new (Date)
 +
 +
(Name)
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:: pbd in Gst-vector
 +
 +
|}
 +
PCR-Mixture for one Reaction:
 +
 +
For a 50 µl reaction use
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>0
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5x Phusion Buffer
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Primer
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P93
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer dw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P94
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Template DNA
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Phusion (add in the end)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|}
 +
What program do you use?
 +
 +
First 25 cycles touchdown 63°C -0,3°C per cycle
 +
 +
next 10 cycles touchdown 72°C -0,3°C per cycle
 +
 +
 +
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 GFP-pbd-PCR
 +
 +
stored in
 +
 +
I will digest it with bamH I and Xho and ligate it to a GST-vector.

Revision as of 15:40, 31 August 2011

Contents

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME


blue light receptor

Miniprep

Investigators: Sandra

Miniprep of:

  • ♥-A3 Not 1
  • ♥-A3 noT 2
  • ♥-A3 noT 3

On the plates with tetracyclin nothing grew.


Testdigest

Investigators: Sandra

Testdigest of minipreps.

Digested with EcoRI and PstI.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

Ligation


Name: Sophie Date: 31.01.11
Continue from Date: 30.08.11 Name: Sophie

Experiment: Digestion

Project Name: inducible promoter for pbd

Procedure


PCR tube:

total volume 20 μl


  1. add H2O (17 μl -X-Y-Z)
  2. add 2 μl Ligase Buffer 10x
  3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
  4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
  5. Add 1 μl T4-DNA Ligase
  6. Incubate 10-30 min at room temperature
  7. heat for 20 minutes at 80°C
  8. 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 S54 both
Y insert 2 ε 5
Z vector psb1C3
H2O

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.


See Digestion...

Transformation


Name:Sophie Date: 31.08.11
Continue from Date: 31.08.11 Name: Sophie

Experiment: Ligation

Project Name: inducible promoter for pbd

Procedure


  1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
  2. thaw cells on ice 20 minutes
  3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
  4. Incubate for 30 minutes on ice
  5. Heat at 42°C for 60 sec
  6. Incubate on ice for 5 minutes
  7. Add 200 μl LB Broth
  8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
  9. 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: S54-ε 5-C3 1:1 / 1:3

stored in incubator on Cm plates

PCR


Name: Sophie


Date: 31.08.11
Continue from Experiment new (Date)

(Name)

Project Name:: pbd in Gst-vector

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 P93
2.5µl Primer dw P94
1µl dNTPs of Template DNA
1µl DNA-Template
0.5 µl Phusion (add in the end)

What program do you use?

First 25 cycles touchdown 63°C -0,3°C per cycle

next 10 cycles touchdown 72°C -0,3°C per cycle


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 GFP-pbd-PCR

stored in

I will digest it with bamH I and Xho and ligate it to a GST-vector.

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/31_August"