Team:Goettingen/NoteBook w1

From 2013.igem.org

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<h2>June 7th, Friday</h2>
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<span class="date"><img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
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<p class="timeline-title">Plasmid mini-prep for Part1-7</p>
<p class="timeline-title">Plasmid mini-prep for Part1-7</p>
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<div class="timeline-cont">
<b>Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):</b>
<b>Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):</b>
<ul>
<ul>
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<p>100uM stock , for PCR dilute 1:20 in HPLC water.</p>
<p>100uM stock , for PCR dilute 1:20 in HPLC water.</p>
<p>Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR.</p>
<p>Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR.</p>
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<h2>June 6th, Thursday</h2>
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<span class="date">06th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
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<div class="cont">
<p class="timeline-title">Pick the colonies of part1-7</p>
<p class="timeline-title">Pick the colonies of part1-7</p>
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<b>Media preparation</b>
<b>Media preparation</b>
<p>1000ml LB+Amp Solid medium => about 50 Plates(with black code)</p>
<p>1000ml LB+Amp Solid medium => about 50 Plates(with black code)</p>
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<b>Pick the colonies of Part1-7</b>
<b>Pick the colonies of Part1-7</b>
<p>4ml LB with antibiotics, overnight culture for mini-prep[marked with C1]</p>
<p>4ml LB with antibiotics, overnight culture for mini-prep[marked with C1]</p>
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<p>Spread backup plates:marked with C1,C2,C3 for each part.</p>
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<p>Backup plates:marked with C1,C2,C3 for each part.</p>
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<h2>June 5th, Wednesday</h2>
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<p class="timeline-title">Preparation of the medium, antibioticks, Transformation.</p>
<p class="timeline-title">Preparation of the medium, antibioticks, Transformation.</p>
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<b>Preparation of Antibiotic Stocks</b>
<b>Preparation of Antibiotic Stocks</b>
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<tr><td>BBa_QO3121</td><td>  P5 20 N</td></tr>
<tr><td>BBa_QO3121</td><td>  P5 20 N</td></tr>
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<h2>June 4th, Tuesday</h2>
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<span class="date">04th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
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<div class="cont">
<p class="timeline-title">Find the correct DNA sequence of DarR and primer design</p>
<p class="timeline-title">Find the correct DNA sequence of DarR and primer design</p>
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<tr style="background = #eee"><th>Code Name</th><th>  Description</th></tr>
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<tr><th>Code Name</th><th>  Description</th></tr>
<tr><td>iGEM_32</td><td>  Forward primer for DarR ORF amplification, with biobrick prefix</td></tr>
<tr><td>iGEM_32</td><td>  Forward primer for DarR ORF amplification, with biobrick prefix</td></tr>
<tr><td>iGEM_33</td><td>  Reverse primer for DarR ORF amplification, with biobrick sufix</td></tr>
<tr><td>iGEM_33</td><td>  Reverse primer for DarR ORF amplification, with biobrick sufix</td></tr>
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<tr><td>iGEM_35</td><td>  Reverse primer for DarR ORF PCR amplification sequencing.</td></tr>
<tr><td>iGEM_35</td><td>  Reverse primer for DarR ORF PCR amplification sequencing.</td></tr>
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Revision as of 07:09, 18 August 2013

Plasmid mini-prep for Part1-7

Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):
  • 900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)
  • vortex
  • store at – 70 °C (red box)
Plasmid Mini-Preparation of parts 1 - 7:

ON cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol for purification of high copy plasmids

Step 5: recommended washing of silica membrane with buffer AW was performed

Step 7: Elution with buffer AE pre-heated to 50 – 60 °C (in future: DON’T elute with this buffer, use pre-heated HPLC-H2O instead! No one knows what’s inside the buffer and its components (EDTA?) could interfere with sequencing and other reactions)


NanoDrop – Plasmid concentrations

Part Numberc(DNA)[ng/μl]A260/A280A260/A230
184.61.942.18
279.81.942.10
3151.01.892.24
429.51.912.07
5154.91.882.25
688.91.922.08
75.914.081.87

Stored in red box at - 20°C

Primers arrived!

iGEM_32 ~ 37: dissolved in HPLC water, stored at -20oC in our box

100uM stock , for PCR dilute 1:20 in HPLC water.

Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR.


06th

Pick the colonies of part1-7

Media preparation

1000ml LB+Amp Solid medium => about 50 Plates(with black code)

Transformation
Entry NumerLocation on the kit
BBa_B0034P5 2 M
Pick the colonies of Part1-7

4ml LB with antibiotics, overnight culture for mini-prep[marked with C1]

Backup plates:marked with C1,C2,C3 for each part.

05th

Preparation of the medium, antibioticks, Transformation.

Preparation of Antibiotic Stocks
  • 1000x Ampicillin 10 1mL Stocks (EPs in the red box in -20 freezer)
  • 1000x Chloramphenicol 10mL Stock (Falcon in Freezer)
Media preparation
  • 250ml*4 LB media with Cm
  • 250ml*1 LB media with Amp
Primer design
Code Name Description
iGEM_36 DarR operator sequence + biobrick prefix
iGEM_37 DarR operator sequence + biobrick sufix

Transformation
Biobrick entry numberMark
BBa_J23117 part 1
BBa_J23116 part 2
BBa_J23110 part 3
BBa_J23118 part 4
BBa_J61101 part 5
BBa_BE0240-Cm part 6 -- Cm
BBa_B0015 part 7 -- Cm
BBa_B0034 part 8
Resuspended but not transformed
Entry number location on kit
BBa_E0204-Amp P5 12 M
BBa_QO3121 P5 20 N
04th

Find the correct DNA sequence of DarR and primer design

Code Name Description
iGEM_32 Forward primer for DarR ORF amplification, with biobrick prefix
iGEM_33 Reverse primer for DarR ORF amplification, with biobrick sufix
iGEM_34 Forward primer for DarR ORF PCR amplification sequencing.
iGEM_35 Reverse primer for DarR ORF PCR amplification sequencing.

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