Difference between revisions of "Team:Marburg/Labbook/CDI"
Sascha Grobe (Talk | contribs) |
Sascha Grobe (Talk | contribs) |
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</table> | </table> | ||
| + | <p> | ||
| + | Turned out that the template was wrong. Inoculated 3 ml LB+chloramphenicol for overnight culture of correct Curly clone (clone No. 3) for Miniprep tomorrow. | ||
| + | <br> | ||
| + | |||
| + | <h1>15/05/22</h1> | ||
| + | |||
| + | <h2>PCR, Gibson Assembly and Transformation </h2> | ||
| + | <p> | ||
| + | Miniprep of 1.5 ml of Curly clone No. 3 according to Thermo Scientific Protocol. | ||
| + | <br> | ||
| + | PCR with primers ICD014 and ICD015 and freshly prepped Curly plasmid to amplify the backbone for the CDI plasmid (pICD001). | ||
| + | Reaction mix and PCR program see 15-05-21. | ||
| + | <br> | ||
| + | Analytical gel: Correct size! (3391 bp) | ||
| + | |||
| + | <figure style="text-align:center;"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/c/c4/MR_pic_CDI_22.5.1.png" width="200" alt="ILS" /> | ||
| + | <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"> | ||
| + | <b>Figure 1:</b> | ||
| + | Ladder, - , PCR | ||
| + | |||
| + | </figcaption> | ||
| + | </figure> | ||
| + | Proceeded with PCR clean up with subsequent Nanodrop measurement: | ||
| + | <br> | ||
| + | Gibson assembly of Curly plasmid/ICD014+015 PCR fragment and CDI operon (EC93 genomic DNA with ICD001 +008) | ||
| + | G.A. reaction mix: | ||
| + | |||
| + | <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;"> | ||
| + | <tr style="background-color:#f66300;"> | ||
| + | <td>Gibson mix </td> | ||
| + | <td> volume </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>Insert</td> | ||
| + | <td class='col'>2.65ul (= 151ng = 0.02 pmol) </td> | ||
| + | |||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>Backbone</td> | ||
| + | <td class='col'>0.54ul (= 45ng = 0.02 pmol) </td> | ||
| + | |||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>G.A. master mix </td> | ||
| + | <td class='col'>10 uL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>ddH2O</td> | ||
| + | <td class='col'>6.81 uL</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | G.A program: | ||
| + | <br> | ||
| + | <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;"> | ||
| + | <tr style="background-color:#f66300;"> | ||
| + | <td>temperature </td> | ||
| + | <td> time </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>50 °C</td> | ||
| + | <td class='col'>15 min</td> | ||
| + | |||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class='col'>16 °C</td> | ||
| + | <td class='col'>store </td> | ||
| + | |||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | Electroporation of Gibson assembly mix with NEB turbo <br> | ||
| + | • Thaw NEB turbo cells on ice <br> | ||
| + | • Dilute G.A. mix 1:3 (5ul mix + 10ul ddH2O)<br> | ||
| + | • Add 2ul of dilution to 25ul of NEB turbo cells <br> | ||
| + | • Transfer the cells to electroporation cuvette <br> | ||
| + | • Electroporate and add 975ul SOC medium immediately <br> | ||
| + | • Recover 1h at 37°C <br> | ||
| + | • Spin down briefly and discard supernatant <br> | ||
| + | • Plate on chloramphenicol plate <br> | ||
| + | |||
| + | <h1>15/05/26</h1> | ||
| + | |||
| + | <h2>PCR, Gibson Assembly and Transformation </h2> | ||
| + | <p> | ||
| + | |||
| + | On 15-05-22 DpnI digestion has been forgotten, so the transformed cells probably carry the Curly plasmid instead of the Gibson assembly product. So we repeated the procedure including DpnI digestion. <br> | ||
| + | PCR of Curly backbone: See 15-05-21 <br> | ||
| + | DpnI digestion program:<br> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
<img src="https://static.igem.org/mediawiki/2015/f/f3/MR_pic_NUTRInitybig.png" class="imgcenter;" style="width:200px;"> | <img src="https://static.igem.org/mediawiki/2015/f/f3/MR_pic_NUTRInitybig.png" class="imgcenter;" style="width:200px;"> | ||
Revision as of 21:19, 12 September 2015
15/05/18
PCR
| PCR | PCR Mix 1 | PCR Mix 2 |
| GXL buffer | 10 uL | 10 uL |
| dNTPs (from GXL kit | 4 uL | 4 uL |
| GXL polymerase | 2uL | 2uL |
| ICD001 (10 uM) | 0.5uL | 1uL |
| ICD007/ICD008 (10 uM) | 0.5uL | 1uL |
| EC93 genomic DNA (15 ng/uL) | 0.5 uL | 1 uL |
| ddH2O | 32.5uL | 29.5uL |
| Program | time |
| Start cycle (30x) | |
| 98 °C | 10 s |
| 60 °C | 15 s |
| 68 °C | 2 min |
| close cycle | |
| 8 °C | store |
Ran of analytical gel. No bands were observed.
15/05/19
PCR
Bands visible! Lanes 1 and 2 right of the ladder should have a 10506 bp band. Lanes 3 and 4 right of the ladder should have a 11414 bp band (gel description should read EC93 instead of EC90).
PCR cleanup (according to Thermo Scientific protocol), elution in 21 ul.
Concentrations according to Nanodrop: EC93 w/ 001+007: 92 ng/ul; EC93 w/ 001+008: 57 ng/ul
15/05/21
PCR
Primers ICD014 and ICD015 arrived! These will be used to amplify the Pick up backbone with overlaps for the full-length Cdi operon. PCR should yield a 3391 bp fragment.
| PCR | PCR Mix 1 |
| GXL buffer | 10 uL |
| dNTPs (from GXL kit | 4 uL |
| GXL polymerase | 2uL |
| ICD0014 (10 uM) | 1 uL |
| ICD0015 (10 uM) | 1uL |
| 1:10 diluted plasmid X | 1 uL |
| ddH2O | 31 uL |
| Program | time |
| Start cycle (30x) | |
| 98 °C | 10 s |
| 60 °C | 15 s |
| 68 °C | 35 s |
| close cycle | |
| 8 °C | store |
Turned out that the template was wrong. Inoculated 3 ml LB+chloramphenicol for overnight culture of correct Curly clone (clone No. 3) for Miniprep tomorrow.
15/05/22
PCR, Gibson Assembly and Transformation
Miniprep of 1.5 ml of Curly clone No. 3 according to Thermo Scientific Protocol.
PCR with primers ICD014 and ICD015 and freshly prepped Curly plasmid to amplify the backbone for the CDI plasmid (pICD001).
Reaction mix and PCR program see 15-05-21.
Analytical gel: Correct size! (3391 bp)
Gibson assembly of Curly plasmid/ICD014+015 PCR fragment and CDI operon (EC93 genomic DNA with ICD001 +008)
G.A. reaction mix:
| Gibson mix | volume |
| Insert | 2.65ul (= 151ng = 0.02 pmol) |
| Backbone | 0.54ul (= 45ng = 0.02 pmol) |
| G.A. master mix | 10 uL |
| ddH2O | 6.81 uL |
| temperature | time |
| 50 °C | 15 min |
| 16 °C | store |
• Thaw NEB turbo cells on ice
• Dilute G.A. mix 1:3 (5ul mix + 10ul ddH2O)
• Add 2ul of dilution to 25ul of NEB turbo cells
• Transfer the cells to electroporation cuvette
• Electroporate and add 975ul SOC medium immediately
• Recover 1h at 37°C
• Spin down briefly and discard supernatant
• Plate on chloramphenicol plate
15/05/26
PCR, Gibson Assembly and Transformation
On 15-05-22 DpnI digestion has been forgotten, so the transformed cells probably carry the Curly plasmid instead of the Gibson assembly product. So we repeated the procedure including DpnI digestion.
PCR of Curly backbone: See 15-05-21
DpnI digestion program:
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