Difference between revisions of "Team:Munich/T4 HOC.html"

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PCR with T4_HOC_F (T4 template) , T4_HOC_His6_R (T4 template) on T4 phages.
 
PCR with T4_HOC_F (T4 template) , T4_HOC_His6_R (T4 template) on T4 phages.
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<figure class="figure">
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  <img src="https://static.igem.org/mediawiki/2018/7/70/T--munich--WL3_Brigit_1.png " class="figure-img img-fluid rounded" alt=" ">
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  <figcaption class="figure-caption">Afterwards Agarose gel, if successful gel extraction</figcaption>
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  </figure>
 
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<td>Notes:</td>
 
<td>Notes:</td>
 
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No notes.  
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<figure class="figure">
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  <img src="https://static.igem.org/mediawiki/2018/4/4b/T--munich--WL3_Brigit_2.png " class="figure-img img-fluid rounded" alt=" ">
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  <figcaption class="figure-caption">Afterwards Agarose gel</figcaption>
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  </figure>
 
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psb1c3_t7 template <br>
 
psb1c3_t7 template <br>
 
pSB1C3_T7_YPET REV (psb1c3_t7 template)
 
pSB1C3_T7_YPET REV (psb1c3_t7 template)
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<figure class="figure">
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  <img src="https://static.igem.org/mediawiki/2018/1/13/T--munich--WL3_Brigit_3.png " class="figure-img img-fluid rounded" alt=" ">
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  <figcaption class="figure-caption"></figcaption>
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  </figure>
  
 
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Elimination of the N-terminal His-tag from the plamid <br> psb1c3_T7_YFP_HOC_His with psb1c3_T7_HisKO_R and psb1c3_T7_HisKO_F
 
Elimination of the N-terminal His-tag from the plamid <br> psb1c3_T7_YFP_HOC_His with psb1c3_T7_HisKO_R and psb1c3_T7_HisKO_F
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<figure class="figure">
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  <img src="https://static.igem.org/mediawiki/2018/8/87/T--munich--WL3_Brigit_4.png " class="figure-img img-fluid rounded" alt=" ">
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  <figcaption class="figure-caption"></figcaption>
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<td>Results:</td>
 
<td>Results:</td>
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<td>Results:</td>
 
<td>Results:</td>
 
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PICTUREEEEEEEEEEEEEEEEEEEEEEEEEEE
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<figure class="figure">
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  <img src="https://static.igem.org/mediawiki/2018/d/d1/T--munich--WL3_Brigit_5.png " class="figure-img img-fluid rounded" alt=" ">
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  <figcaption class="figure-caption"></figcaption>
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  </figure>
 
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<td>Results:</td>
 
<td>Results:</td>
 
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HERE
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<figure class="figure">
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  <img src="https://static.igem.org/mediawiki/2018/a/a4/T--munich--WL3_Brigit_6.png " class="figure-img img-fluid rounded" alt=" ">
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  <figcaption class="figure-caption">Positive clone after sequencing, went on with transformation into BL21(E3)STAR</figcaption>
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  </figure>
 
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Revision as of 00:23, 18 October 2018

PCR- Primer test for Gibson Cloning of N-HOC-His-C in psb1c3_t7_ypet

2018/07/02
Participants: Keno Eilers
Protocol: PCR
Notes: Generated his- hoc: T4_HOC_F (T4 template), T4_HOC_His6_R (T4 template)
Insert His6 HOC FWD (HOC-His)
Insert His6 HOC REV (HOC-His) --> generates gibson overlap
pSB1C3_T7_YPET FWD (psb1c3_t7 template)
pSB1C3_T7_YPET REV (psb1c3_t7 template) --> generates gibson overlap
Afterwards always Agarose gel: 2ul of T4 Phage stock solution for PCR 200ng psb1c3_T7_ypet.
Results: Worked out.

Primer test for N-HOC-TEV-His-C

2018/07/03
Participants: Keno Eilers
Protocol: PCR
Notes: T4_HOC_TEV_His_F_short (T4 template)
T4_HOC_TEV_His_R (T4 template) --> generates overhang
HOC-TEV_His FWD (HOC-TEV-His)
HOC-TEV-His REV (HOC-TEV-His) --> generates gibson overlap
psb1c3_T7_YPET_HOC_His6 FWD
psb1c3_T7_YPET_HOC_His6 REV --> generates gibson overlap
Afterwards always Agarose gel: 2ul of T4 Phage stock solution for PCR 200ng psb1c3_T7_ypet.
Results: Worked out.

Primer test for His-KO from psb1c3_T7_YFP_HOC_His

2018/07/04
Participants: Keno Eilers
Protocol: PCR
Notes: psb1c3_T7_HisKO_R (psb1c3_T7_YFP_HOC_His template)
psb1c3_T7_HisKO_F (psb1c3_T7_YFP_HOC_His template) --> His KO of the N-terminus (psb1c3_T7_YFP_HOC_HisKO)
psb1c3_T7_HisÜB_F (psb1c3_T7_YFP_HOC_HisKO template)
psb1c3_T7_HisÜB_R(psb1c3_T7_YFP_HOC_HisKO template) --> His insertion on the N-terminus
Afterwards Agarose gel, 2ul of T4 Phage stock solution for PCR. 200ng psb1c3_T7_ypet.
Results: Worked out.

PCR for Gibson Cloning of N-HOC-His-C in psb1c3_t7_ypet

2018/07/22
Participants: Keno Eilers
Protocol: PCR
Notes: PCR with T4_HOC_F (T4 template) , T4_HOC_His6_R (T4 template) on T4 phages.
Afterwards Agarose gel, if successful gel extraction
Results: Worked out

PCR with "Insert His6 HOC FWD (HOC-His)", "Insert His6 HOC REV (HOC-His)" on purified PCR product from the previous step

2018/07/24 -2018/07/25
Participants: Keno Eilers
Protocol: PCR
Notes:
Afterwards Agarose gel
Results: Worked, afterwards PCR Cleanup.

Preperation of the corresponding backbone with pSB1C3_T7_YPET FWD

2018/07/25-2018/07/26
Participants: Keno Eilers
Protocol: PCR
Notes: psb1c3_t7 template
pSB1C3_T7_YPET REV (psb1c3_t7 template)
Results: PCR Cleanup after successful PCR

Gibson Ligation

2018/07/30
Participants: Keno Eilers
Protocol: Dpn1 digest, PCR cleanup, Gibson Assembly
Notes: 0ng backbone, 100ng insert.
Results: Worked out.

Transformation

2018/07/31-2018/08/03
Participants: Keno Eilers
Protocol: NAME, Chem. Tarnsformation, sequencing,overnight culture, miniprep, elekto-comp. transformation
Notes: No notes.
Results: Worked out.

PCR for Gibson Cloning of psb1c3_T7_His_YFO_HOC

2018/08/06- 2018/08/07
Participants: Keno Eilers
Protocol: PCR
Notes: Elimination of the N-terminal His-tag from the plamid
psb1c3_T7_YFP_HOC_His with psb1c3_T7_HisKO_R and psb1c3_T7_HisKO_F
Results: Worked out.

psb1C3 Hoc-His preparation for Transformation

2018/08/08
Participants: Keno Eilers
Protocol: NAME, Dpn1 digest, agarose gel, pcr cleanup kit, Gibson assembly
Notes: No notes.
Results: Worked out, transformation has been done.

psb1C3_yfp_Hoc-His transformation

2018/08/07-2018/08/09
Participants: Keno Eilers
Protocol: NAME, Chem. Trafo, pver night culture, mini-prep
Notes: Plasmid has been sequenced.
Results: Worked out --> have been transformed into BL21(E3)STAR

Test Expression of YFP-HOC-His

2018/08/13-2018/08/14
Participants: Keno Eilers
Protocol: NAME, Protein Expression
Notes: No notes.
Results: Samples have been used for timepoint determinations.

Timepoint determinations through SDS-Page

2018/08/20
Participants: Keno Eilers
Protocol: NAME, SDS-Page gel
Notes: No notes.
Results:

Western Plot with yfp_his_hoc

2018/08/22-2018/08/23
Participants: Keno Eilers
Protocol: NAME, SDS-gel, Westernplot
Notes: No notes.
Results: Did not work out.

YFP_his_hoc vector 2 -DNA phosphorylation

2018/09/14
Participants: Keno Eilers
Protocol: NAME, DNA Posphorilation , PCR cleanup kit
Notes: No notes.
Results: DNA concentation to low, no ligation possible.

psb1C3_yfp_Hoc-His transformation

2018/09/014-2018/09/15
Participants: Keno Eilers
Protocol: NAME, Chem Trafo, over night culture, miniprep, sequencing
Notes: No notes.
Results: Overnight culture was pinned down, pelet was frozen away --> Monday miniprep.

Miniprep

2018/09/17
Participants: Keno Eilers
Protocol: NAME, Miniprep,1% agarose gel
Notes: concenration determination by band intensity.
Results:
Positive clone after sequencing, went on with transformation into BL21(E3)STAR

Transformation from positive clone in DH5alpha and BL21(E3)STAR

2018/09/18 – 2018/09/21
Participants: Keno Eilers, Brigit Tunaj
Protocol: NAME, Chem. Transformation, miniprep
Notes: No notes.
Results: Worked out--> now expression of yfp_his_hoc.

Protein expression of yfp_his_hoc

2018/09/24-2018/09/25
Participants: Keno Eilers
Protocol: NAME, Protein expression
Notes: HERE
Results: Sample were checked with fluorescence microscopy --> yfp is still active

Test of yfp_his_hoc with Western Blot

2018/09/27
Participants: Keno Eilers
Protocol: NAME, Western Blot
Notes: No notes.
Results: Western Blot was positive --> can be used for phage Assembly.