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| Line 46: |
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| | <div class="labbook"> | | <div class="labbook"> |
| | </div> | | </div> |
| − |
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| − | =Week 7 (June 27th - July 3rd)=
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| − | <div class="week" id="WWeek_7">
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| − |
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| − | =='''Monday, June 27th'''==
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| − | === Sequencing of P67 (EGFR-Signalpeptid) ===
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| − |
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| − | '''Investigator: Niklas'''
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| − |
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| − | '''Procedure:'''
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| − |
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| − | Sequencing batch was prepared after manufacturer's protocol. (15 µl of plasmid DNA (100 ng) and 2 µl sequencing primer (VF2))
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| − |
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| − | FR11326586
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| − | </div>
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| − |
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| − | <div>
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| − |
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| − | <div>
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| − | === Repetition of Quick-Change PCR of P3 (pASK + SAm1) ===
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| − |
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| − | '''Investigator: Luisa'''
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| − |
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| − | '''Procedure:'''
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| − |
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| − | * The QC-PCR was performed according the SOP.
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| − |
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| − | * Reaction Mix:
| |
| − | <table cellspacing="0" border="1">
| |
| − | <tr>
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| − | <td><b>volume</b>
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| − | </td><td><b>reagent</b>
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| − | </td></tr>
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| − | <tr>
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| − | <td>1,25 µl
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| − | </td><td>Primer O21
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| − | </td></tr>
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| − | <tr>
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| − | <td>1,25 µl
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| − | </td><td>Primer O22
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| − | <tr>
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| − | <td>1 µl
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| − | </td><td> dNTP-mix
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| − | </td></tr>
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| − | <tr>
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| − | <td> 5 µl
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| − | </td><td>Pfu-Ultra-II reaction buffer
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| − | <tr>
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| − | <td>1 µl
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| − | </td><td> template DNA (1:10 dilution of p3)
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| − | </td></tr>
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| − | <tr>
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| − | <td> 0,5 µl
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| − | </td><td>Pfu-Ultra-II Polymerase
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| − | <tr>
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| − | <td>40,5 µl
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| − | </td><td> ddH2O
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| − | </td></tr></table>
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| − |
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| − | * Digestion of PCR-Product with DpnI for 1h at 37°C.
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| − |
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| − | * Transformation of 10µl into component E.coli XL-1-blue, according to SOP (1h incubation at 37°C necessary despite AmpR).
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| − | </div>
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| − |
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| − | === PCR of Genesynthesis 3 and 4 ===
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| − |
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| − | '''Investigator: Luisa'''
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| − |
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| − | '''Aim of Experiment: Amplification of Genesynthesis 3 (contains BAP and IGKappa) and 4 (contains A3C5-tag and BM40)'''
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| − |
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| − | '''Procedure:'''
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| − |
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| − | * The PCR was performed according the SOP.
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| − |
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| − | * Reaction Mix:
| |
| − | <table cellspacing="0" border="1">
| |
| − | <tr>
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| − | <td><b>volume</b>
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| − | </td><td><b>reagent</b>
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| − | </td></tr>
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| − | <tr>
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| − | <td> 2,5 µl
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| − | </td><td>Primer VF2
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| − | </td></tr>
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| − | <tr>
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| − | <td> 2,5 µl
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| − | </td><td>Primer VR2
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| − | <tr>
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| − | <td> 1 µl
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| − | </td><td> dNTP-mix
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| − | </td></tr>
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| − | <tr>
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| − | <td> 10 µl
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| − | </td><td> Q5 Polymerase reaction buffer
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| − | <tr>
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| − | <td>1 µl
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| − | </td><td> template DNA (1:10 dilution of p3)
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| − | </td></tr>
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| − | <tr>
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| − | <td> 0,5 µl
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| − | </td><td> Q5-Polymerase
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| − | <tr>
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| − | <td> 18 µl
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| − | </td><td> ddH2O
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| − | </td></tr></table>
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| − |
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| − | *Setup: iGEM_standard (Promega-cycler)
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| − | <table cellspacing="0" border="1">
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| − | <tr>
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| − | <td><b>temperature</b>
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| − | </td><td><b>time</b>
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| − | </td></tr>
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| − | <tr>
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| − | <td> 98°C
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| − | </td><td> 2min
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| − | </td></tr>
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| − | <tr>
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| − | <td> 98°C
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| − | </td><td> 10sec
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| − | <tr>
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| − | <td> 66°C
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| − | </td><td> 30sec
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| − | </td></tr>
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| − | <tr>
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| − | <td> 72°C
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| − | </td><td> 30sec
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| − | <tr>
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| − | <td> 72°C
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| − | </td><td> 2min
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| − | </td></tr>
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| − | <tr>
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| − | <td> 4°C
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| − | </td><td> hold
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| − | </td></tr></table>
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| − |
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| − | * the batches were then purified using the Quiagen PCR-Purification Kit.
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| − |
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| − | </div>
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| − |
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| − | === Analytical digestion and gelelectrophoresis of P88 , P89 and P90 ===
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| − |
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| − | '''Investigator: Niklas'''
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| − |
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| − | '''Aim of experiment: Analytical digestion and gelelectrophoresis of P88 (pASK75 + Streptactin, former F58), P89 (CMV + CD4, former F65) and P90 (CMV + EGFR-signal-peptide, former F66)'''
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| − |
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| − | '''Procedure:'''
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| − | * Batches for analytical digestions:
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| − |
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| − | P88: EcoRI
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| − |
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| − | P89: EcoRI and PstI
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| − |
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| − | P90: EcoRI
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| − | {|cellspacing="0" border="1"
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| − | |'''volume'''
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| − | |'''reagent'''
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| − | |-
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| − | |5,8/2,3/1,8 µl
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| − | |Plasmid DNA (P88/P89/P90)
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| − | |-
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| − | |1 µl
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| − | |CutSmart buffer (10x)
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| − | |-
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| − | |0.5 µl
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| − | |EcoRI-HF(10 U/µl)/ PstI
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| − | |-
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| − | |required amount for total volume of 10 µl
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| − | |ddH2O
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| − | |}
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| − |
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| − | [[File:Muc16_P88-90_NA.JPG]]
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| − |
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| − | === Ligation of F67 and F71, Transformation of ''E. coli'' XL1 blue afterwards ===
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| − |
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| − | '''Investigator: Niklas'''
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| − |
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| − | '''Aim of the experiment:''' Ligation of F67 (BirA) and F71 (empty pSB1C3), Transformation of ''E. coli'' XL1 blue afterwards.
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| − |
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| − | '''Procedure:'''
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| − | {|cellspacing="0" border="1"
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| − | |'''volume'''
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| − | |'''reagent'''
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| − | |-
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| − | |2,4 µl
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| − | |Vektor
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| − | |-
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| − | |7,6 µl
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| − | |Insert
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| − | |-
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| − | |2 µl
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| − | |10X DNA-Ligase-buffer
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| − | |-
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| − | |1 µl
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| − | |T4-Ligase
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| − | |-
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| − | |7 µl
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| − | |ddH<sub>2</sub>O
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| − | |-
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| − | |=20 µl
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| − | |'''TOTAL'''
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| − | |}
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| − |
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| − | * CaCl2 competent ''E. coli'' XL1-Blue cells were taken out of stock in -80 °C freezer and were gently thawed on ice.
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| − |
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| − | * 7 µl of DNA was added to 100 µl of competent cells and gently mixed.
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| − |
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| − | * 30 min incubation on ice
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| − |
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| − | * 5 min. heat shock at 37 °C
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| − |
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| − | * Adding of 750 µl LB-medium to each tube.
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| − |
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| − | * Incubation for 1 hour at 37 °C
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| − |
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| − | * The cell suspension was plated on Cam-plates (inclusive rescue plate) and incubated over night at 37 °C in the cell-culture shaker.
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| − |
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| − | * next step: analytic digestion of transformation was successful
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| − |
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| − | </div>
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| − |
| |
| − | <div>
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| − |
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| − | === Digestion of PCR on genesynthesis 3 and 4, and pSB1C3 ===
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| − |
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| − | '''Investigator: Luisa'''
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| − |
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| − | '''Aim of experiment: Division of Leptin, IGKappa, A3C5, BM40 and BAP using SapI, HindIII, XbaI, AgeI for both batches.'''
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| − |
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| − | '''Procedure:'''
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| − | * Batches for analytical digestions:
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| − |
| |
| − | {|cellspacing="0" border="1"
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| − | |'''volume'''
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| − | |'''reagent'''
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| − | |-
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| − | |1 µl each
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| − | |enzyme (SapI, HindIII, XbaI, AgeI)
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| − | |-
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| − | |5 µl
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| − | |CutSmart buffer (10x)
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| − | |-
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| − | |41 µl
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| − | |DNA (purified PCR-products of GSY3 and 4)
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| − | |-
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| − | |}
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| − |
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| − | * Additionally 10µg of the vector P74 was digested with XbaI and AgeI in 100µl batch (2µl of each enzyme, 10µl of Cut-Smart buffer). Digestion was performed over night and purified via gelelectrophoresis and gelextraction according to the manufacturer's protocoll. --> Now labeled F80.
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| − |
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| − | </div>
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| − |
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| − | === Analytical digestion and gelelectrophoresis of P80 , P78 and P85 ===
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| − |
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| − | '''Investigator: Julian'''
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| − |
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| − | '''Aim of experiment: Analytical digestion and gelelectrophoresis of P80 (mRuby3), P78 (NanoLuc) and P85(Strep-Tag)'''
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| − |
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| − | '''Procedure:'''
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| − | * Batches for analytical digestions:
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| − |
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| − | *P80 and P78: EcoRI and AgeI
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| − |
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| − | *P85: EcoRI and NgoMIV
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| − |
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| − | {|cellspacing="0" border="1"
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| − | |'''volume'''
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| − | |'''reagent'''
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| − | |-
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| − | |10 µl
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| − | |Plasmid DNA
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| − | |-
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| − | |32 µl
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| − | |ddH2O
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| − | |-
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| − | |5 µl
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| − | |CutSmart buffer (10x)
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| − | |-
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| − | |1.5 µl
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| − | |each enzyme(10 U/µl)/ PstI
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| − | |-
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| − | |50 µl
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| − | |'''TOTAL'''
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| − | |}
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| − |
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| − | [[File:Muc16_.JPG]]
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| − |
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| − | === Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3 with Strep-Tag) and Transformation into ''E. coli'' XL1 blue ===
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| − |
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| − | '''Investigator: Luisa'''
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| − |
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| − | '''Aim of the experiment:''' Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3+Strep-Tag), Transformation of ''E. coli'' XL1 blue afterwards.
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| − |
| |
| − | '''Procedure:'''
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| − | {|cellspacing="0" border="1"
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| − | |'''volume'''
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| − | |'''reagent'''
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| − | |-
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| − | |4,3µl(for F75), 8,2µl (for F76)
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| − | |Vector
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| − | |-
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| − | |12,7µl (F75), 8,8 (F76)
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| − | |Insert
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| − | |-
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| − | |2 µl
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| − | |10X DNA-Ligase-buffer
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| − | |-
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| − | |1 µl
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| − | |T4-Ligase
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| − | |-
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| − | |=20 µl
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| − | |'''TOTAL'''
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| − | |}
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| − |
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| − | *Ligation was incubated at RT for 1,5h.
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| − |
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| − | * CaCl2 competent ''E. coli'' XL1-Blue cells were taken out of stock in -80 °C freezer and were gently thawed on ice.
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| − |
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| − | * 7 µl of DNA was added to 100 µl of competent cells and gently mixed.
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| − |
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| − | * 15 min incubation on ice
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| − |
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| − | * 5 min. heat shock at 37 °C
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| − |
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| − | * Adding of 950 µl LB-medium to each tube.
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| − |
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| − | * Incubation for 1 hour at 37 °C
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| − |
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| − | * The cell suspension was plated on Cam-plates (inclusive rescue plate) and incubated over night at 37 °C in the incubator.
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| − |
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| − | === Chemical biotinylation of BSA ===
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| − |
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| − | '''Investigator: Niklas'''
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| − |
| |
| − | '''Procedure:'''
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| − |
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| − | * BSA was chemically biotinylated with a 20x and 40x molar excess:
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| − |
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| − | * 10 ml of 100 mM borate buffer with 50 mM NaCl (pH 8.85)
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| − |
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| − | * dissolve BSA (10 mg/ml)
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| − |
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| − | * Add biotin-NHS-ester: 20,5 mg for 40x molar excess
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| − |
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| − | * reaction over night
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| − | </div>
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| − |
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| − | =='''Tuesday, June 28th'''==
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| − | </div>
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| − |
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| − | =='''Wednesday, June 29th'''==
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| − | </div>
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| − |
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| − | <!--- this closes the week -->
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| − | </div>
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| − | <!--- ^^^^ this closes the week -->
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| − | <!--- PLEASE DO NOT TOUCH !!!! -->
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| − | <!--- PLEASE DO NOT TOUCH !!!! -->
| |
