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Team:Goettingen/NoteBook w1
From 2013.igem.org
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| + | <h2>June 7th, Friday</h2> | ||
| + | <p class="timeline-title">Plasmid mini-prep for Part1-7</p> | ||
| + | <b>Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):</b> | ||
| + | <ul> | ||
| + | <li>900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)</li> | ||
| + | <li>vortex</li> | ||
| + | <li>store at – 70 °C (red box)</li> | ||
| + | <br /> | ||
| + | <b>Plasmid Mini-Preparation of parts 1 - 7:</b> | ||
| + | <p>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</p> | ||
| + | <p>Step 5: recommended washing of silica membrane with buffer AW was performed</p> | ||
| + | <p>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)</p> | ||
| + | <br / > | ||
| + | <p>NanoDrop – Plasmid concentrations</p> | ||
| + | <table cellspacing="0"> | ||
| + | <tr><th>Part Number</th><th>c(DNA)[ng/μl]</th><th>A<sub>260</sub>/A<sub>280</sub></th><th>A<sub>260</sub>/A<sub>230</sub></th> | ||
| + | <tr><td>1</td><td>84.6</td><td>1.94</td><td>2.18</td></tr> | ||
| + | <tr><td>2</td><td>79.8</td><td>1.94</td><td>2.10</td></tr> | ||
| + | <tr><td>3</td><td>151.0</td><td>1.89</td><td>2.24</td></tr> | ||
| + | <tr><td>4</td><td>29.5</td><td>1.91</td><td>2.07</td></tr> | ||
| + | <tr><td>5</td><td>154.9</td><td>1.88</td><td>2.25</td></tr> | ||
| + | <tr><td>6</td><td>88.9</td><td>1.92</td><td>2.08</td></tr> | ||
| + | <tr><td>7</td><td>5.9</td><td>14.08</td><td>1.87</td></tr> | ||
| + | </table> | ||
| + | <p>Stored in red box at - 20°C</p> | ||
| + | |||
| + | <br /> | ||
<h2>June 6th, Thursday</h2> | <h2>June 6th, Thursday</h2> | ||
<p class="timeline-title">Pick the colonies of part1-7</p> | <p class="timeline-title">Pick the colonies of part1-7</p> | ||
Revision as of 06:08, 17 August 2013
June 7th, Friday
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)
- 1000x Ampicillin 10 1mL Stocks (EPs in the red box in -20 freezer)
- 1000x Chloramphenicol 10mL Stock (Falcon in Freezer)
- 250ml*4 LB media with Cm
- 250ml*1 LB media with Amp
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 Number | c(DNA)[ng/μl] | A260/A280 | A260/A230 |
|---|---|---|---|
| 1 | 84.6 | 1.94 | 2.18 |
| 2 | 79.8 | 1.94 | 2.10 |
| 3 | 151.0 | 1.89 | 2.24 |
| 4 | 29.5 | 1.91 | 2.07 |
| 5 | 154.9 | 1.88 | 2.25 |
| 6 | 88.9 | 1.92 | 2.08 |
| 7 | 5.9 | 14.08 | 1.87 |
Stored in red box at - 20°C
June 6th, Thursday
Pick the colonies of part1-7
Media preparation1000ml LB+Amp Solid medium => about 50 Plates(with black code)
Transformation| Entry Numer | Location on the kit | |||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| BBa_B0034 | P5 2 M | Pick the colonies of Part1-7
| Code Name | Description |
|---|---|
| iGEM_36 | DarR operator sequence + biobrick prefix |
| iGEM_37 | DarR operator sequence + biobrick sufix |
Transformation
| Biobrick entry number | Mark |
|---|---|
| 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 |
June 4th, Tuesday
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. |
