Team:Freiburg/Notebook/17 August

From 2011.igem.org

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Name of the ligation-product: ♥-A3
Name of the ligation-product: ♥-A3
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|}Transformation''' '''
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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: 17.08.11
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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 Date: 17.08.11 Name: Sophie
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Experiment: Ligation
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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: new 3A assembly with Amp-vector, Blue light receptor
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|}
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Procedure
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# take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
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# 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
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# 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
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'''Documentation:'''
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Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.
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 +
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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: ♥-A3
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stored in incubator on Amp-plates
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vector: psBA3
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inserts: LovTAP, NOT-Gate
|}
|}

Revision as of 15:31, 17 August 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

Contents

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?

  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 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:

  • 15ng/μl

stored in undigested miniprep box II


Testdigest

Investigators: Sandra

Testdigest of Miniprep product.

Enzymes:

  • EcoRI
  • PstI

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


  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 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


  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: ♥-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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