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

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<td>
 
<td>
 
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a>,  
 
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a>,  
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href="HERE" target="_blank">NAME</a>,
+
href="https://static.igem.org/mediawiki/2018/a/ab/T--Munich--WL1_Sequencing_sample_preparation.pdf" target="_blank">Sequenzing</a>,  
−
href="HERE" target="_blank">NAME</a>,  
+
href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep</a>,  
−
href="HERE" target="_blank">NAME</a>,  
+
href="https://static.igem.org/mediawiki/2018/c/ca/T--Munich--WL1_Electrocompetent_transformation.pdf" target="_blank">Electrocompetent Transformation</a>
−
href="HERE" target="_blank">NAME</a>,
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−
Chem. Tarnsformation, sequencing,overnight culture, miniprep, elekto-comp. transformation
 
  
 
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<td>Protocol:</td>
 
<td>Protocol:</td>
 
<td>
 
<td>
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<a href="HERE" target="_blank">NAME</a>,  
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<a href="https://static.igem.org/mediawiki/2018/4/46/T--Munich--Protocol_DNA_methylation.pdf" target="_blank">DpnI digest</a>,  
−
 
+
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose Gel</a>,  
−
Dpn1 digest, agarose gel, pcr cleanup kit, Gibson assembly  
+
<a href="https://international.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug#Product%20Information" target="_blank">PCR Cleanup Kit</a>,  
 +
<a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson assembly</a>
  
 
</td>
 
</td>
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<td>Protocol:</td>
 
<td>Protocol:</td>
 
<td>
 
<td>
−
<a href="HERE" target="_blank">NAME</a>,  
+
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical Transformation</a>,  
−
 
+
<a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep</a>
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Chem. Trafo, pver night culture,  mini-prep
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<td>Protocol:</td>
 
<td>Protocol:</td>
 
<td>
 
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<a href="HERE" target="_blank">NAME</a>,
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<a href="https://static.igem.org/mediawiki/2018/5/53/T--Munich--TX-TL10.pdf" target="_blank">TX-TL</a>
  
 
Protein Expression
 
Protein Expression
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<td>Protocol:</td>
 
<td>Protocol:</td>
 
<td>
 
<td>
−
<a href="HERE" target="_blank">NAME</a>,  
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<a href="https://international.neb.com/protocols/2014/10/24/rapid-pngase-f-by-sds-page-protocol-p0710" target="_blank">SDS-Page</a>,  
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+
−
SDS-Page gel
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<td>Protocol:</td>
 
<td>Protocol:</td>
 
<td>
 
<td>
−
<a href="HERE" target="_blank">NAME</a>,  
+
<a href="https://international.neb.com/protocols/2014/10/24/rapid-pngase-f-by-sds-page-protocol-p0710" target="_blank">SDS-gel</a>,  
−
 
+
<a href="https://international.neb.com/protocols/2016/03/23/western-blot" target="_blank">Western blot</a>
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SDS-gel, Westernplot
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</td>
 
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<td>Protocol:</td>
 
<td>Protocol:</td>
 
<td>
 
<td>
−
<a href="HERE" target="_blank">NAME</a>,
+
<a href="https://international.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug#Product%20Information" target="_blank">PCR cleanup kit</a>
 +
 
  
−
DNA Posphorilation , PCR cleanup kit
 
  
 
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<td>Protocol:</td>
 
<td>Protocol:</td>
 
<td>
 
<td>
−
<a href="HERE" target="_blank">NAME</a>,  
+
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical Transformation</a>,  
 +
<a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep</a>,
 +
 
 +
<a href="https://static.igem.org/mediawiki/2018/a/ab/T--Munich--WL1_Sequencing_sample_preparation.pdf" target="_blank">sequencing</a>
  
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Chem Trafo, over night culture, miniprep, sequencing
 
  
 
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<td>Protocol:</td>
 
<td>Protocol:</td>
 
<td>
 
<td>
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<a href="HERE" target="_blank">NAME</a>,  
+
<a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>
  
−
Miniprep,1%  agarose gel
 
  
 
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<td>Protocol:</td>
 
<td>
 
<td>
−
<a href="HERE" target="_blank">NAME</a>,  
+
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical Transformation</a>,  
 +
<a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep</a>
 +
 
  
−
Chem. Transformation, miniprep
 
  
 
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<td>Protocol:</td>
 
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<td>
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<a href="HERE" target="_blank">NAME</a>,
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<a href="https://static.igem.org/mediawiki/2018/5/53/T--Munich--TX-TL10.pdf" target="_blank">TX-TL</a>
  
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Protein expression
 
  
 
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<a href="HERE" target="_blank">NAME</a>,
 
  
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Western Blot
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<a href="https://international.neb.com/protocols/2016/03/23/western-blot" target="_blank">Western blot</a>
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Revision as of 02:57, 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: Chemical transformation, href="https://static.igem.org/mediawiki/2018/a/ab/T--Munich--WL1_Sequencing_sample_preparation.pdf" target="_blank">Sequenzing, href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep, href="https://static.igem.org/mediawiki/2018/c/ca/T--Munich--WL1_Electrocompetent_transformation.pdf" target="_blank">Electrocompetent 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: DpnI 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: Chemical Transformation, Miniprep
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: TX-TL 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: SDS-Page,
Notes: No notes.
Results:

Western Plot with yfp_his_hoc

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

YFP_his_hoc Vector 2 -DNA Phosphorylation

2018/09/14
Participants: Keno Eilers
Protocol: 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: Chemical Transformation, 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: Miniprep, 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: Chemical 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: TX-TL
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: Western blot
Notes: No notes.
Results: Western Blot was positive --> can be used for phage Assembly.