Team:Munich/T4 HOC.html

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, Sequenzing, Miniprep, 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.