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)
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)
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)
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
Results:
PCR Cleanup after successful PCR
Gibson Ligation
2018/07/30
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
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.
