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.
|
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: |
No notes.
|
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
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: |
PICTUREEEEEEEEEEEEEEEEEEEEEEEEEEE
|
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: |
HERE
|
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.
|
