Team:Freiburg/Notebook/25 July

From 2011.igem.org

(Difference between revisions)
(→NAME OF YOUR EXPERIMENT)
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'''PCR'''
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{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Sophie
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 25.07.11
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|-
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| 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 (Date)
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(Name): Commons
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|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: more linearized backbones (4 different vectors)
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|}
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PCR-Mixture for one Reaction:
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For a 50 µl reaction use
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{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5µl
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| 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
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5x Phusion Buffer
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Primer
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| SB-prep-3P-1
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer dw
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| SB-prep-2Ea
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Template DNA
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PSB 1 A3
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PSB 1 C3
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PSB 1 K3
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PSB 1 T3
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl
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| 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)
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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|}
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What program do you use?
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# 2Min 94°C
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# 30s 94°C
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# 30s 55°C
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# 3min 72°C
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# 10min 72°C
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step 2,3 and 4 in 35 cycles
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To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
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How did you label the PCR-Product, where is it stored and what do you do next?
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Labeled PSB 1 A3, PSB 1 C3, PSB 1 K3 and PSB 1 T3 stored in -20°C in last drawer
==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
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'''Investigators:NAME'''
'''Investigators:NAME'''
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==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==

Revision as of 14:11, 25 July 2011

PCR


Name: Sophie


Date: 25.07.11
Continue from Experiment (Date)

(Name): Commons

Project Name: more linearized backbones (4 different vectors)

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 A3

PSB 1 C3

PSB 1 K3

PSB 1 T3

0.5 µl Phusion (add in the end)

What program do you use?

  1. 2Min 94°C
  2. 30s 94°C
  3. 30s 55°C
  4. 3min 72°C
  5. 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 A3, PSB 1 C3, PSB 1 K3 and PSB 1 T3 stored in -20°C in last drawer

Contents

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

NAME OF YOUR EXPERIMENT

Gibson NOT-Gate

Investigators:

Sophie

Transformation


Name: Sophie Date: 25.07.11
Continue from Date Name

Experiment

Project Name: Blue light (NOT-Gate)

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.


We need a NOT Gate for our Blue light system. In this experiment I transformed E.coli with the Part Bba_Q04400 from the iGEM distribution kit. The vector is pSBK3 with Kanamycin resistance. The strain ist named S 45 and will be plated out. The plates will be stored in the incubator.


PCR


Name: Sophie


Date: 25.07.11
Continue from Experiment: new experiment. We need NOT-Gate and LovTAP with Gibson-overhangs, so 2 PCRs are made
Project Name: Blue Light

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 NOT_G_up

LOV_G_up

2.5µl Primer dw NOT_G_dw

LOV_G_dw

1µl dNTPs of Template DNA
1µl DNA-Template S35 (BBa_K322999)

S45 (BBa_Q04400)

0.5 µl Phusion (add in the end)

What program do you use?

67c auf 70c


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?

I labelled it with a heart and a star and stored it in the minipreps 3 box.

I will do Gibson assemblz of the two parts next.

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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