Team:Hannover/Notebook

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Notebook

date	coworkers	lab	activity	short summary
22.09.2014	Lisa, Kathi	IPG	insertion of rcf21	colony-PCR –> only negative controls –> consultation of TR → modification of the protocol
19.09.2014	Fabian	IPG	preparation of large scale E. coli cultures	For mass spectometric analyses, large amounts of bacteria with T4MBP are needed. Therefore 2 l of Origami 2 with pASK_T4MBP and 0,25 mM cadmium were prepared. The chassis Origami 2 with and without cadmium are used as controls (2 l each). Induction of proteinexpression with anhydrotetracycline. Growing at 25 °C for 3 d to reach a high OD
19.09.2014	Fabian	IPG	transformation of pASK (no insert) into Origami 2	Analyses showed that a comparable expression system for pASK_T4MBP is needed. Therefore chemical competent cells were made. Transformation via heat shock, incubation over weekend at 16 °C
19.09.2014	Kathi	IPG	insertion of rcf21	plasmid isolation from ONC; digestion of pORE-E3 (without XhoI and BglII-site) with MluI and BamHI; Annealing two primers (3 and 4) to build the rcf; phosphorylation of primer construct; dephosphorylation of digested vector. ligation of vector and insert; transformation of XL1-Blue Competent Cells. incubation over night (25 °C)
19.09.2014	Steffen	IPG	plasmidpreparation and sequencing	-plasmidprep of ONC (red/white colony pSB1C3) and colony T4MBP -sequencing with primer 16, T4MBP sequenced with primer 16/17
18.09.2014	Fabian	IPG	physiological test of T4MBP activity	Origami 2 with pASK_T4MBP at different cadmium concentrations (1-0 mM) were analyzed. At 0.2 mM high grow rates were observed. In comparison to Origami 2 without T4MBP no significant effect of TMBP was seen. Problem: induction of proteinexpression reduces growth rates. Another inducible protein in Origami 2 is needed for comparison.
18.09.2014	Kathi	IPG	insertion of rcf21	digestion of pORE-E3 (without XhoI- and BglII-site). There was no DNA → inoculation of an ONC
18.09.2014	Steffen	IPG	colony-PCR	-colony-PCR using E. coli colonies (16.09.2014). Using Primer 16 and 17. Detection of 1 positive clone TMBP -red and white colonies growed (pSB1C3) colony-PCR
17.09.2014	Fabian, Steffen	IPG	immunostain	Immunostaining blotted PVDF-membranes. Using Strep-Tag antibody and His-Tag antibody. Anti-Strep shows specific signal at 37 kD. Anti-His show signal at 37 kD as well. His was used as positive controll. Anti-Strep obviously works. Wanted protein is found in inclusion bodies and supernatant.
17.09.2014	Kathi, Björn	IPG	insertion of rcf21	colony-PCR with primer 1261 and 488 → all 60 colonies were negative → digestion went probably wrong. isolation of plasmid form another overnight culture via handprep
16.09.2014	Fabian, Steffen	IPG	SDS-PAGE, Western Blot	Testing new Strep-Tag antibody. Using pASK_T4MBP in Origami2 to produce protein. Harvesting protein via ultrasound sonification. Testing protein pellet and supernatant. Runs discontinuous SDS-PAGE. Blotting porteins on PVDF. Checking transfer via Ponceau-stain. Blocking membrane over night with Roti-Block
16.09.2014	Kathi, Björn	IPG	insertion of rcf21	plasmid isolation from ONC; digestion of pORE-E3 (without XhoI and BglII-site) with MluI and BamHI; Annealing two primers (3 and 4) to build the rcf; phosphorylation of primer construct; dephosphorylation of digested vector. ligation of vektor and insert; transformation of XL1-Blue Competent Cells.
16.09.2014	Steffen	IPG	saving linearized pSB1C3 into E. coli and transformation of E. coli	-1.) ligation reactions with/without ligase 2.) ligation for 1h at room temperature 5.) Transformation of XL1-Blue Competent Cells via heat-shock using 10 µl ligation reaction 6.) selection on chloramphenicol –1.)Transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction 11.09.2014 2.) selection on chloramphenicol
15.09.2014	Kathi, Björn	IPG	insertion of rcf21	digestion of pORE-E3 (without XhoI and BglII-site). There was no DNA → inoculation of an overnight culture
15.09.2014	Fabian	Botany	selection of transformed A. thaliana seeds	Sterilized the harvested A. thaliana seeds (09.09.2014) with ethanol. Plated the seed on Murashige & Skoog media with 2 % sucrose and 15 µg/ml phosphinothricin as selection. Stored them for 2 days at 4 °C. Put at 20 °C until germination
12.09.2014	Steffen	IPG	colony-PCR and sequencing results	-Colony-PCR using E. coli colonies (11.09.2014). Using Primer 16 and 17. Detection of 0 positive clone Expansin/TMBP -results of expansin and CBD are positive
11.09.2014	Anke, Lisa	IPG	plasmidpreparation and sequencing
11.09.2014	Steffen, Anke	IPG	growth curves of Origami 2 pASK, cloning T4MBP, CBD into pSB1C3 (shipping vector) and plasmidpreparation/sequencing	-Growth of Origami 2 pASK in media containing five different cadmium concentration, measured the OD600 over a period of 7 hours (7 measurements) -1.) PCR-Purification of PCR-products (10.09.2014) 2.) Restriction digest of PCR-product and pSB1C3 with FastDigest EcoRI and PstI 3.) Purification of digested products 4.) Ligation for 1 h at room temperature 5.) Transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction 6.) selection on chloramphenicol -plasmidprep (before Glystocks prepared) overnigth culture positive colonies Expansin/CBD–>sequencing with primer 16
10.09.2014	Steffen	IPG	colony-PCR	-Colony-PCR using E. coli colonies (09.09.2014). Using Primer 16 and 17. Detection of 1 positive clone Expansin -overnight culture positive colonies CBD/Expansin
09.09.2014	Fabian, Anke	Biophysics	confocal microscopy	Microscopy of transformed B. sinuspersici and N. tabacum from 04.09.2014.
09.09.2014	Steffen	IPG	colony-PCR	-Colony-PCR usingE. coli colonies (08.09.2014). Using Primer 16 and 17. Detection of 1 positive clone CBD -1.)Transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction 5.09.2014 2.) selection on chloramphenicol
08.09.2014	Fabian, Steffen	Botany	harvesting of transgenic seeds from A. thaliana	Harvesting of mature seeds of transformed plants (transformation date: 31.07.2014)
08.09.2014	Steffen	IPG	transformation of E.coli Xl1-blue with pSB1C3	1.)Transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction 5.09.2014 2.) selection on chloramphenicol
05.09.2014	Steffen	IPG	cloning T4MBP, Expansin, CBD into pSB1C3 (shipping vector)	FastDigest enzymes tested (expansin digested/not digested run on 2.5% gel)–>showed that digestion was positive 1.) PCR-Purification of PCR-product (4.09.2014) 2.) Restriction digest of PCR-product and pSB1C3 with FastDigest EcoRI and PstI 3.) Purification of digested products 4.) Ligation for 1 h at room temperature
04.09.2014	Fabian	Botany	transient plant transformation	A. tumefaciens mediated transformation of B. sinuspersici and N. tabacum. Used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plmamembranemarker. Using leaf infiltration for tobacco and vacuum ilfiltration for Bienertia.
03.09.2014	Steffen	IPG	colony-PCR	Colony-PCR using E. coli colonies (02.09.2014). Using Primer 16 and 17. Detection of 0 positive clones
02.09.2014	Steffen	IPG	cloning T4MBP, Expansin, CBD into pSB1C3 (shipping vector)	1.) PCR-Purification of PCR-product (1.09.2014) 2.) Restriction digest of PCR-product and pSB1C3 with NEB EcoRI and PstI 3.) Purification of digested products 4.) Ligation for 1 h at room temperature 5.) Transformation of XL1-Blue Competent Cells via heat-shock and selection on chloramphenicol
01.09.2014	Fabian, Steffen, Anke, Andi	Biophysics	confocal microscopy	Microscopy of transformed B. sinuspersici and N. tabacum from 29.08.2014.
29.08.2014	Fabian	Botany	transient plant transformation	A. tumefaciens mediated transformation of B. sinuspersici and N. tabacum. Used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plmamembranemarker. Using leaf infiltration for tobacco and vacuum infiltration for Bienertia.
27.08.2014	Melanie, Lisa	IPG	plasmidpreparation and sequencing.v1	plasmidprep with 12 colonies, double digest (XhoI and NcoI), all probes on gel, 7 might be without BglII AND XhoI → sequencing with primer ??? (IPG) positive → pORE_E3 2x35S (without T4MPB) and without BglII- and XhoI-site
26.08.2014	Melanie	IPG	ONC of 13 colonies (12+1 pos. control)	several colonies on each plate (including pos. control), pick of 3 to 4 per plate, ONC
25.08.2014	Melanie, Lisa	IPG	ligation, transformation of E. coli with pORE_E3 2x35S (without T4MBP and without BglII-site) ?without? XhoI-site	ligation, transformation of XL1-Blue Competent Cells via heat-shock, 3 x plating on Can
22.08.2014	Melanie	IPG	digest of pORE_E3 2x35S (without T4MBP and without BglII-site) with XhoI, proofreading PCR	digest of pORE_E3 2x35S (without T4MPB and without BglII-site) with XhoI for 1h 37 °C showed complete digestion; proofreading-PCR to avoid A-Tailing (Phusion)
22.08.2014	Fabian	Botany	colony-PCR	Colony-PCR using E. coli colonies (20.08.2014). Using Primer 11 (AG Offermann) and 1261. Detection of 15 positive clones. Using 3 colonies for ONC
21.08.2014	Fabian	Botany	cloning Expa~GFP~CBD into pORE	1.) PCR-Purification of PCR-product (19.08.2014) 2.) Restriction digest of PCR-product and pORE E3 2x35S with BamHI and MluI 3.) Gelelectrophoresis 4.) Gelextraction of digested vector and PCR-product 5.) Ligation for 1h 6.) Transformation of XL1-Blue Competent Cells via heat-shock and selection on kanamycin
20.08.2014	Melanie	IPG	sequencing.v3 (new primer)	sequencing with primer 1356 was positive → BglII-site destroyed
20.08.2014	Kathi, Björn	IPG	insertion of T4MBP in pASK	sequencing confirms the insertion of T4-MBP in pASK
19.08.2014	Fabian	Botany	Phusion PCR	Phusion PCR using Expa~GFP~CBD-sequence from EMP as template
19.08.2014	Melanie	IPG	sequencing.v2 (same primer)	sequencing with primer 1261 (IPG) again stopped 10 bp before BglII-site, no definite result → again
18.08.2014	Kathi, Björn	IPG	insertion of T4MBP in pASK	sequencing of pASK with T4MBP not long enough
15.08.2014	Lisa	IPG	plasmidpreparation and sequencing.v1	plasmidprep with 10 colonies, digest with BglI (twice), poor results, only 2 probes on gel, 1 might be without BglII → sequencing with primer 1261 (IPG) of this probe stopped 10 bp before BglII-site, no definite result → again
14.08.2014	Kathi	IPG	insertion of T4MBP in pASK	plasmid isolation → shipping to Seqlab
13.08.2014	Kathi	IPG	insertion of T4MBP in pASK	colony PCR with primers 729 and 734 → one positive colony → ONC
13.08.2014	Melanie, Lisa	IPG	ONC of 10 colonies (9+1 pos. control)	several colonies on each plate (including pos. control), pick of 3 per plate, ONC
12.08.2014	Melanie	IPG	transformation of E. coli with pORE_E3 2x35S (without T4MBP) ?without? BglII	via heat-shock, 3 x plating on Can
12.08.2014	Kathi, Björn	IPG	insertion of T4MBP in pASK	Preparations of XL1-Blue Competent Cells; transformation of XL1-Blue Competent Cells with ligation mixture
11.08.2014	Kathi	IPG	insertion T4MBP in pASK	amplification of T4MBP with primers 8 and 9. Addition of EcoRI und NcoI sites via Primers; puriciation of PCR reaction mixture via kit; digestion of pASK and amplificat with EcoRI and NcoI; ligation over night (16 h, 16°C)
11.08.2014	Melanie, Lisa	IPG	digest of pORE_E3 2x35S (without T4MBP) with BglII, proofreading PCR, ligation	digest of pORE_E3 2x35S (without T4MPB) with BglII for 1h 37 °C showed complete digestion; proofreading-PCR to avoid A-Tailing (Phusion), ligation
11.08.2014	Kathi, Björn	IPG	insertion of T4MBP in pASK	amplification of T4MBP by adding EcoRI and NcoI sites with the primers 8 and 9; digestion of amplificat and pASK vector with EcoRI and NcoI; simulatnous dephosphorlyation of pASK, ligation of pASK and T4-MBP (16 h, 16 °C)
10.08.2014	Andi	IPG	immunostain.v2	see 07.08; still no difference between control and samples; 9 min are enough for the final incubation of substrate buffer
09.08.2014	Andi	IPG	colony-PCR, SDS-PAGE.v2	with oligonucleotides 729, 734 (BCH) & Ta = 48 °C (1:30 Min)
08.08.2014	Andi	IPG	plasmid-isolation, pASK-traformation.v2	Isolation of pORE-E3 for future cloning and EMP_in_pMA_Colony1 for sequencing. Transformation of Kathis and Björns ligation-product in freshly prepared heatshock competent BL21 (DE3) pLyss cells.
08.08.2014	Kathi, Björn	IPG	insertion of T4MBP in pASK	isolation of pASK from overnight culture
07.08.2014	Andi	IPG	immunostain, colony-PCR, ONC	polyclonal anti-Flag-antibody were used as primary, and anti-rabbit alkaline phosphatase as secondary antibody (both 1:2000 diluted). after incubating the substrate buffer for 13 min, the same signals (all over the lanes; very unspecific) were observed in the samples and control, respectively. We were not sure, if problems in the sample handling are the cause for that, and suggest a repetition. Although colony-PCR (F1+R1) confirmed the insertion of GFP, another primer set (BCH: 1101+625) targeting the backbone of pMA did not bind in the colony, but showed a not expected amplificate length of ~1200 bp in the negative control (expected for pMA: ~1600 bp) ONC of pORE-E3,
07.08.2014	Kathi, Björn	IPG	insertion of T4MBP in pASK	only colonies on positive control → again inoculation of overnight culture
06.08.2014	Anke, Andi	IPG	SDS-PAGE, Coomassie-stain & Blotting	The volume of cellulose-bound protein samples were reduced by direct application of „Polyethylenglykol 6000“ on top of the tube. All samples were loaded into a 12 % SDS-PAGE.
06.08.2014	Kathi, Björn	IPG	insertion of T4MBP in pASK	Amplification of CDS (without HIS-TAG) with primers 8 and 9 (Botany) via Phusion. Purification of the product with „Wizard-PCR and Gel Kit) Double digest of PCR product and pASK vector with EcoRI and NcoI for 30 min. Purification of both samples. Poor results. Ligation of insert and pASK for 2 h (22 °C); transformation over night (37 °C)
05.08.2014	Andi	IPG	cellulose-bound-protein GFP_in_pMA_EMP2	Dialyzing four times to remove Urea from the cellulose samples (1x overnight, 2x during the day, 1x overnight). Repetion of EMP-PCR(2) reaction mix no. 2 (protocol of Anke). After an agarosegel confirmed the correct fragment length, the reaction mix was purfied using the „Wizard-Kit“ (BCH), phosphorylated and ligated overnight according to Ankes protocol.
05.08.2014	Kathi, Björn	IPG	insertion of T4MBP in pASK	Isolation of Plasmid (pORE_E3_2x35S_Expa_T4MBP_CBD) from E. coli overnight culture via Plasmidprep. MiniKit (Firma?). Amplification of CDS (without HIS-TAG) with Primers 8 and 9 (Botany) via Phusion. → did not work → again
04.08.2014	Anke, Andi	IPG	isolation of transient expressed protein from N. tabacum	Isolation of cellulose-bound and unbound protein from N. tabaccum. The material was cooled by liquid nitrogen, automatically macerated by „Precellys“ and resuspended in 1 x SDS-sample buffer. Per plant one sample was taken. The debris-pellet from centrifugation was washed with ddH20 two times and incubated in 1 ml 8 M Urea overnight.
31.07.2014	Steffen, Fabian	Botany	floral dip transformation of A. thaliana	Transformation of A. thaliana via Floral Dip by using A. tumefaciens as vector. Construct: pORE_E3_2x35S_Expa_T4MBP_CBD
31.07.2014	Anke, Björn	IPG	transient transformation of N. tabacum with pORE_E3_2x35S_Expa_TMBP_CBD	transient Transformation of 5 N. tabacum plants with pORE_E3_2x35S_TMBP in GV1301 and 4 plants without GV1301. Using 1ml / leave and two leaves / plant.
25.07.2014	Anke	IPG	colony-PCR of EMP2-transformation	Colony-PCR with primers 1011 and 625 (IPG) and 10 new colonies. Poor result…
23.07.2014	Anke	IPG	colony-PCR of EMP2-transformation	Colony-PCR with primers 1011 and 625 (IPG). Poor result….
22.07.2014	Anke	IPG	transformation of E. coli with EMP2	Transformation of XL1-Blue Competent Cells
21.07.2014	Anke, Andi	IPG	EMP 2 GfP	Screening the functional amount of megaprimer with and without F1: 20 ng Megaprimer + F1 + R2
21.07.2014	Fabian	Botany	colony-PCR of Agor-Trafo	Colony-PCR with primer 11 (AG Offermann and 1484). Poor result, proabably of too high annealing temperature. New colony-PCR with 11 and 1261. Prepare 2 days cultures for Arabidopsis transformation
18.07.2014	Anke	IPG	EMP 1 GfP	EMP1 with F1, R2 and GFP-vector to genererate Megaprimer. Result is fine.
18.07.2014	Fabian	Botany	transformation of Agrobacterium with pORE_E3_2x35S_Expa_TMBP_CBD	Using electrocompetent GV3101 cells.
17.07.2014	Steffen, Fabian	Botany	plasmidpreparation and sequencing	Using ONC of E. coli with pORE and insert. Plasmidprep via Thermo Kit. Sequencing from both directions with primer 11 (AG Offermann) and 1261. Poor results for reverse direction. Resequencing of reverse sequence with 1484
15.07.2014	Steffen, Fabian	Botany	colony-PCR of pORE with Insert	Taq-PCR of 27 colonies using primer 11 of AG Offermann (binds to 35S) and 1012. Poor result, wrong reverse primer used. Doing new colony PCR with other reverse primer 1261 (binds to NOS-terminator. Result is fine. ONC
14.07.2014	Steffen, Fabian	Botany	cloning of CDS (Expa_T4MBP_CBD) into pORE2x35S	1. PCR-Purification of Phusion-PCR (11.7) to get rid of disturbing enzymes. 2.) double digest of purified PCR-product and vector with MluI and BamHI for 1 h. 3.) Preparative Gelelctrophoresis. Cutting out specific bands. 4.) Gelextraction. 5.) Ligation of digested insert and vector for 1 h. 6.) Transformation of chemical competent E. coli DH5alpha with ligated DNA via heatshock
11.07.2014	Fabian	Botany	amplification of CDS from polyprotein	Phusion-PCR. Using Geneart-Shipping vector with insert as template. Primers 106 and 859
03.07.2014	Kathi	IPG	negative colony-PCR	(continued by Fabian and Steffen)
02.07.2014	Kathi	IPG	repetition of day 27.06.2014: Transformation of XL1blue with T4MBP in pORE-E3 with 35S-Promotor
01.07.2014	Kathi	IPG	due to unsuccessful transformation repetition of day 26.06.2014: ligation of T4MBP with pORE-E3 with 35S-Promotor	Digestion of pASK with T4MBP and pORE-E3 with 2x35S promotor with MluI and BamHI, overnight ligation
30.06.2014	Andi	IPG	sequencing Bielefelder-CBDs	Sequencing (Seqlab/ Microsynth) was done with primer no. 611 (GCTGGCCTTTTGCTCAGATGTTCTTTCCTGCGTTATC) and revealed that the obtained sequencing result matched the given sequence online
30.06.2014	Kathi	IPG	negative colony-PCR
27.06.2014	Kathi	IPG	transformation of XL1-Blue with T4MBP in pORE-E3 with 35S-Promotor
26.06.2014	Kathi	IPG	ligation of T4MBP with pORE-E3 with 35S-Promotor	Digestion of pASK with T4MBP and pORE-E3 with 2x35S promotor with Mlu1 and BamHI, overnight ligation
26.06.2014	Kathi	IPG	plasmidpreparation of pASK	Digestion of pASK with T4MBP and pORE-E3 with 2x35S promotor with Mlu1 and BamHI, overnight ligation
25.06.2014	Kathi	IPG	colony-PCR of transformated XL1blue with T4MBP in pASK	Primer for colony PCR: T7 and BackSeq-pGII
24.06.2014	Kathi	IPG	transformation to back up the synthesised T4MBP located in pASK vector	Transformation of XL1-Blue Competent Cells
24.06.2014	Melanie	IPG	linearization of Bielefelder-CBDs	linearizing BBa_K863101 & BBa_K863111
24.06.2014	Melanie, Andi	IPG	isolation of Bielelfelder-CBDs	Isolating BBa_K863101 & BBa_K863111 with PeqGOLD Plasmid Miniprep Kit by PeqLab GmbH and selfmade solutions
20.06.2014	Andi	IPG	ONC of Bielefelder-CBDs	Culturing XL1 containg pSB1C3 plasmid with parts BBa_K863101 & BBa_K863111 in 10 ml LB medium and 10 µl Chloramphenicol
19.06.2014	Fabian	Botany	plasmidpreparation	Preparation of the 3 E. coli clones which were expected to have the right insert (using Thermo Mini-Prep Kit). Plasmids of one all 3 clones were send for sequencing, using Primer 1011 (IPG). Sequencing worked fine. All 3 clones have the insert at the right position
18.06.2014	Fabian	Botany	new colony-PCR	New Colony-PCR for detecting transformed E. coli clones. Using primers 1011 and 1012 (IPG). Detecting 3 positive clones wich were used for over-night cultures
16.06.2014	Fabian	Botany	plasmidpreparation	Preparation of 2E. coli clones wich were expected to have the right insert (using Thermo Mini-Prep Kit). Plasmid of one clone was send for sequencing, using Primer 962 (IPG). Sequencing didn't work (second sequncing trial by seqlab failed as well). Probably Primer 962 isn't working (even without 2x35S insertion sequencing should have work)
13.06.2014	Fabian	Botany	colony-PCR	Colony-PCR for detecting transformed E. coli clones. Using amplification primers 01 and 02 AND 1011 (IPG). Colony-PCR worked more or less, results were interpreted wrongly. False clones were used for over-night cultures
12.06.2014	Fabian	Botany	digest, ligation and transformation of 2x35S into pORE	1) Gelextraction of 2x35S (using Qiagen-Kit). 2) Digest of 2x35S and pORE with XhoI and BamHI (37 °C for 1 h). 3) Gelelectophoresis for separation of cutted pORE, using the large fragment (Gelextraction). 4) Ligation of 2x35S and pORE (using T7-Ligase). 5) Transformation of chemical competent E. coli DH5-Alpha and plating on LB-plates with Kanamycin
10.06.2014	Fabian	Botany	amplification of 2x35S Promotor	Using Primer 01 and 02 to amplify 2x35S promotor from template DNA. Separation of fragments via gelelectophoresis and preparation on fragments from the gel
21.05.2014	Fabian	Botany	BamHI, MluI-digest	Analysing if both Enzymes (BamHI and MluI) cut while double digesting pORE E3
19.05.2014	Anke	IPG	-	Preparing Glystock
19.05.2014	Steffen, Fabian	Botany	-	plasmid isolation pORE-E3
16.05.2014	Steffen, Andi	IPG	„over-weekend“- culture pORE-E3	Culturing JM109 bacteria cells containing pORE-E3 (250 µM from Glystock) in 10 ml LB media including 10 µM Kanamycin (Stock?) over weekend at 27 °C

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-<h3>Notebook</h3><p class="text">Test</p>
+<h3>Notebook</h3>
+<table class="inline">
+	<tbody><tr class="row0">
+		<td class="col0">date </td><td class="col1">coworkers </td><td class="col2">lab </td><td class="col3">activity </td><td class="col4">short summary </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row1">
+		<td class="col0">22.09.2014 </td><td class="col1">Lisa, Kathi </td><td class="col2">IPG </td><td class="col3">insertion of rcf21 </td><td class="col4">colony-PCR &ndash;&gt; only negative controls &ndash;&gt; consultation of TR → modification of the protocol </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">19.09.2014 </td><td class="col1">Fabian </td><td class="col2">IPG </td><td class="col3">preparation of large scale <em>E. coli</em> cultures </td><td class="col4">For mass spectometric analyses, large amounts of bacteria with T4MBP are needed. Therefore 2 l of Origami 2 with pASK_T4MBP and 0,25 mM cadmium were prepared. The chassis Origami 2 with and without cadmium are used as controls (2 l each). Induction of proteinexpression with anhydrotetracycline. Growing at 25 °C for 3 d to reach a high OD </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">19.09.2014 </td><td class="col1">Fabian </td><td class="col2">IPG </td><td class="col3">transformation of pASK (no insert) into Origami 2 </td><td class="col4">Analyses showed that a comparable expression system for pASK_T4MBP is needed. Therefore chemical competent cells were made. Transformation via heat shock, incubation over weekend at 16 °C </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">19.09.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">insertion of rcf21 </td><td class="col4">plasmid isolation from ONC; digestion of pORE-E3 (without XhoI and BglII-site) with MluI and BamHI; Annealing two primers (3 and 4) to build the rcf; phosphorylation of primer construct; dephosphorylation of digested vector. ligation of vector and insert; transformation of XL1-Blue Competent Cells. incubation over night (25 °C) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">19.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">plasmidpreparation and sequencing </td><td class="col4">-plasmidprep of ONC (red/white colony pSB1C3) and colony T4MBP      <br>
+  <br>
+-sequencing with primer 16, T4MBP sequenced with primer 16/17 </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">18.09.2014 </td><td class="col1">Fabian </td><td class="col2">IPG </td><td class="col3">physiological test of T4MBP activity </td><td class="col4">Origami 2 with pASK_T4MBP at different cadmium concentrations (1-0 mM) were analyzed. At 0.2 mM high grow rates were observed. In comparison to Origami 2 without T4MBP no significant effect of TMBP was seen. Problem: induction of proteinexpression reduces growth rates. Another inducible protein in Origami 2 is needed for comparison. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row7">
+		<td class="col0">18.09.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">insertion of rcf21 </td><td class="col4">digestion of pORE-E3 (without XhoI- and BglII-site). There was no DNA → inoculation of an ONC </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row8">
+		<td class="col0">18.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">colony-PCR </td><td class="col4">-colony-PCR using <em>E. coli</em> colonies (16.09.2014). Using Primer 16 and 17. Detection of 1 positive clone TMBP      <br>
+  <br>
+-red and white colonies growed (pSB1C3) colony-PCR </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row9">
+		<td class="col0">17.09.2014 </td><td class="col1">Fabian, Steffen </td><td class="col2">IPG </td><td class="col3">immunostain </td><td class="col4">Immunostaining blotted PVDF-membranes. Using Strep-Tag antibody and His-Tag antibody. Anti-Strep shows specific signal at 37 kD. Anti-His show signal at 37 kD as well. His was used as positive controll. Anti-Strep obviously works. Wanted protein is found in inclusion bodies and supernatant. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row10">
+		<td class="col0">17.09.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of rcf21 </td><td class="col4">colony-PCR with primer 1261 and 488 → all 60 colonies were negative → digestion went probably wrong. isolation of plasmid form another overnight culture via handprep </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row11">
+		<td class="col0">16.09.2014 </td><td class="col1">Fabian, Steffen </td><td class="col2">IPG </td><td class="col3">SDS-PAGE, Western Blot </td><td class="col4">Testing new Strep-Tag antibody. Using pASK_T4MBP in Origami2 to produce protein. Harvesting protein via ultrasound sonification. Testing protein pellet and supernatant. Runs discontinuous SDS-PAGE. Blotting porteins on PVDF. Checking transfer via Ponceau-stain. Blocking membrane over night with Roti-Block </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row12">
+		<td class="col0">16.09.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of rcf21 </td><td class="col4">plasmid isolation from ONC; digestion of pORE-E3 (without XhoI and BglII-site) with MluI and BamHI; Annealing two primers (3 and 4) to build the rcf; phosphorylation of primer construct; dephosphorylation of digested vector. ligation of vektor and insert; transformation of XL1-Blue Competent Cells. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row13">
+		<td class="col0">16.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3 leftalign">saving linearized pSB1C3 into <em>E. coli</em> and transformation of <em>E. coli</em>  </td><td class="col4">-1.) ligation reactions with/without ligase 2.) ligation for 1h at room  temperature 5.) Transformation of XL1-Blue Competent Cells via heat-shock using 10 µl ligation reaction  6.) selection on chloramphenicol    <br>
+  <br>
+&ndash;1.)Transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction 11.09.2014 2.) selection on chloramphenicol </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row14">
+		<td class="col0">15.09.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of rcf21 </td><td class="col4">digestion of pORE-E3 (without XhoI and BglII-site). There was no DNA → inoculation of an overnight culture </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row15">
+		<td class="col0">15.09.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">selection of transformed <em>A. thaliana</em> seeds </td><td class="col4">Sterilized the harvested <em>A. thaliana</em> seeds (09.09.2014) with ethanol. Plated the seed on Murashige &amp; Skoog media with 2 % sucrose and 15 µg/ml phosphinothricin as selection. Stored them for 2 days at 4 °C. Put at 20 °C until germination </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row16">
+		<td class="col0">12.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">colony-PCR and sequencing results </td><td class="col4">-Colony-PCR using <em>E. coli</em> colonies (11.09.2014). Using Primer 16 and 17. Detection of 0 positive clone Expansin/TMBP    <br>
+  <br>
+-results of expansin and CBD are positive </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row17">
+		<td class="col0">11.09.2014 </td><td class="col1">Anke, Lisa </td><td class="col2">IPG </td><td class="col3">plasmidpreparation and sequencing </td><td class="col4 leftalign">  </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row18">
+		<td class="col0">11.09.2014 </td><td class="col1">Steffen, Anke </td><td class="col2">IPG </td><td class="col3">growth curves of Origami 2 pASK, cloning T4MBP, CBD into pSB1C3 (shipping vector) and plasmidpreparation/sequencing </td><td class="col4">-Growth of Origami 2 pASK in media containing five different cadmium concentration, measured the OD600 over a period of 7 hours (7 measurements)    <br>
+  <br>
+-1.) PCR-Purification of PCR-products (10.09.2014) 2.) Restriction digest    of PCR-product and pSB1C3 with FastDigest EcoRI and PstI 3.)  Purification of  digested products 4.)  Ligation for 1 h at room  temperature 5.) Transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction  6.) selection on chloramphenicol       <br>
+ <br>
+-plasmidprep (before Glystocks prepared) overnigth culture positive colonies Expansin/CBD&ndash;&gt;sequencing with primer 16 </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row19">
+		<td class="col0">10.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">colony-PCR </td><td class="col4">-Colony-PCR using <em>E. coli</em> colonies (09.09.2014). Using Primer 16 and 17. Detection of 1 positive clone Expansin    <br>
+  <br>
+-overnight culture positive colonies CBD/Expansin </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row20">
+		<td class="col0">09.09.2014 </td><td class="col1">Fabian, Anke </td><td class="col2">Biophysics </td><td class="col3">confocal microscopy </td><td class="col4">Microscopy of transformed <em>B. sinuspersici</em> and <em>N. tabacum</em> from 04.09.2014. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row21">
+		<td class="col0">09.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">colony-PCR </td><td class="col4">-Colony-PCR using<em>E. coli</em> colonies (08.09.2014). Using Primer 16 and 17. Detection of 1 positive clone CBD    <br>
+  <br>
+-1.)Transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction 5.09.2014 2.) selection on chloramphenicol </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row22">
+		<td class="col0">08.09.2014 </td><td class="col1">Fabian, Steffen </td><td class="col2">Botany </td><td class="col3 leftalign">harvesting of transgenic seeds from <em>A. thaliana</em>  </td><td class="col4">Harvesting of mature seeds of transformed plants (transformation date: 31.07.2014) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row23">
+		<td class="col0">08.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">transformation of E.coli Xl1-blue with pSB1C3 </td><td class="col4">1.)Transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction 5.09.2014 2.) selection on chloramphenicol </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row24">
+		<td class="col0">05.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">cloning T4MBP, Expansin, CBD into pSB1C3 (shipping vector) </td><td class="col4">FastDigest enzymes tested (expansin digested/not digested run on 2.5% gel)&ndash;&gt;showed that digestion was positive      <br>
+  <br>
+.) PCR-Purification of PCR-product (4.09.2014) 2.) Restriction digest   of PCR-product and pSB1C3 with FastDigest EcoRI and PstI 3.) Purification of  digested products 4.)  Ligation for 1 h at room temperature </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row25">
+		<td class="col0">04.09.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">transient plant transformation </td><td class="col4"><em>A. tumefaciens</em> mediated transformation of <em>B. sinuspersici</em> and <em>N. tabacum</em>. Used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plmamembranemarker. Using leaf infiltration for tobacco and vacuum ilfiltration for Bienertia. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row26">
+		<td class="col0">03.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">colony-PCR </td><td class="col4">Colony-PCR using <em>E. coli</em> colonies (02.09.2014). Using Primer 16 and 17. Detection of 0 positive clones </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row27">
+		<td class="col0">02.09.2014 </td><td class="col1">Steffen </td><td class="col2">IPG </td><td class="col3">cloning T4MBP, Expansin, CBD into pSB1C3 (shipping vector) </td><td class="col4">1.) PCR-Purification of PCR-product (1.09.2014) 2.) Restriction digest  of PCR-product and pSB1C3 with NEB EcoRI and PstI 3.) Purification of digested products 4.)  Ligation for 1 h at room temperature 5.) Transformation of XL1-Blue Competent Cells via heat-shock and  selection on chloramphenicol </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row28">
+		<td class="col0">01.09.2014 </td><td class="col1">Fabian, Steffen, Anke, Andi </td><td class="col2">Biophysics </td><td class="col3">confocal microscopy </td><td class="col4">Microscopy of transformed <em>B. sinuspersici</em> and <em>N. tabacum</em> from 29.08.2014. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row29">
+		<td class="col0">29.08.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">transient plant transformation </td><td class="col4"><em>A. tumefaciens</em> mediated transformation of <em>B. sinuspersici</em> and <em>N. tabacum</em>. Used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plmamembranemarker. Using leaf infiltration for tobacco and vacuum infiltration for Bienertia. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row30">
+		<td class="col0">27.08.2014 </td><td class="col1">Melanie, Lisa </td><td class="col2">IPG </td><td class="col3">plasmidpreparation and sequencing.v1 </td><td class="col4">plasmidprep with 12 colonies, double digest (XhoI and NcoI), all probes on gel, 7 might be without BglII AND XhoI → sequencing with primer ??? (IPG) positive → pORE_E3 2x35S (without T4MPB) and without BglII- and XhoI-site </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row31">
+		<td class="col0">26.08.2014 </td><td class="col1">Melanie </td><td class="col2">IPG </td><td class="col3">ONC of 13 colonies (12+1 pos. control) </td><td class="col4">several colonies on each plate (including pos. control), pick of 3 to 4 per plate, ONC </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row32">
+		<td class="col0">25.08.2014 </td><td class="col1">Melanie, Lisa </td><td class="col2">IPG </td><td class="col3">ligation, transformation of <em>E. coli</em> with pORE_E3 2x35S (without T4MBP and without BglII-site) ?without? XhoI-site </td><td class="col4">ligation, transformation of XL1-Blue Competent Cells via heat-shock, 3 x plating on Can </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row33">
+		<td class="col0">22.08.2014 </td><td class="col1">Melanie </td><td class="col2">IPG </td><td class="col3">digest of pORE_E3 2x35S (without T4MBP and without BglII-site) with XhoI, proofreading PCR </td><td class="col4">digest of pORE_E3 2x35S (without T4MPB and without BglII-site) with XhoI for 1h 37 °C showed  complete digestion; proofreading-PCR to avoid A-Tailing (Phusion) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row34">
+		<td class="col0">22.08.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">colony-PCR </td><td class="col4">Colony-PCR using E. coli colonies (20.08.2014). Using Primer 11 (AG Offermann) and 1261. Detection of 15 positive clones. Using 3 colonies for ONC </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row35">
+		<td class="col0">21.08.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">cloning Expa~GFP~CBD into pORE </td><td class="col4">1.) PCR-Purification of PCR-product (19.08.2014) 2.) Restriction digest of PCR-product and pORE E3 2x35S with BamHI and MluI 3.) Gelelectrophoresis 4.) Gelextraction of digested vector and PCR-product 5.) Ligation for 1h 6.) Transformation of XL1-Blue Competent Cells via heat-shock and selection on kanamycin </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row36">
+		<td class="col0">20.08.2014 </td><td class="col1">Melanie </td><td class="col2">IPG </td><td class="col3">sequencing.v3 (new primer) </td><td class="col4">sequencing with primer 1356 was positive   → BglII-site destroyed </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row37">
+		<td class="col0">20.08.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">sequencing confirms the insertion of T4-MBP in pASK </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row38">
+		<td class="col0">19.08.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">Phusion PCR </td><td class="col4">Phusion PCR using Expa~GFP~CBD-sequence from EMP as template </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row39">
+		<td class="col0">19.08.2014 </td><td class="col1">Melanie </td><td class="col2">IPG </td><td class="col3">sequencing.v2 (same primer) </td><td class="col4">sequencing with primer 1261 (IPG) again stopped 10 bp before BglII-site, no definite result  → again </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row40">
+		<td class="col0">18.08.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">sequencing of pASK with T4MBP not long enough </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row41">
+		<td class="col0">15.08.2014 </td><td class="col1">Lisa </td><td class="col2">IPG </td><td class="col3">plasmidpreparation and sequencing.v1 </td><td class="col4">plasmidprep with 10 colonies, digest with BglI (twice), poor results, only 2 probes on gel, 1 might be without BglII → sequencing with primer 1261 (IPG) of this probe stopped 10 bp before BglII-site, no definite result  → again </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row42">
+		<td class="col0">14.08.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">plasmid isolation → shipping to Seqlab </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row43">
+		<td class="col0">13.08.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">colony PCR with primers 729 and 734 → one positive colony → ONC </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row44">
+		<td class="col0">13.08.2014 </td><td class="col1">Melanie, Lisa </td><td class="col2">IPG </td><td class="col3">ONC of 10 colonies (9+1 pos. control) </td><td class="col4">several colonies on each plate (including pos. control), pick of 3 per plate, ONC </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row45">
+		<td class="col0">12.08.2014 </td><td class="col1">Melanie </td><td class="col2">IPG </td><td class="col3">transformation of <em>E. coli </em> with pORE_E3 2x35S (without T4MBP) ?without? BglII </td><td class="col4">via heat-shock, 3 x plating on Can </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row46">
+		<td class="col0">12.08.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">Preparations of XL1-Blue Competent Cells; transformation of XL1-Blue Competent Cells with ligation mixture </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row47">
+		<td class="col0">11.08.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">insertion T4MBP in pASK </td><td class="col4">amplification of T4MBP with primers 8 and 9. Addition of EcoRI und NcoI sites via Primers; puriciation of PCR reaction mixture via kit; digestion of pASK and amplificat with EcoRI and NcoI; ligation over night (16 h, 16°C) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row48">
+		<td class="col0">11.08.2014 </td><td class="col1">Melanie, Lisa </td><td class="col2">IPG </td><td class="col3">digest of pORE_E3 2x35S (without T4MBP) with BglII, proofreading PCR, ligation </td><td class="col4">digest of pORE_E3 2x35S (without T4MPB) with BglII for 1h 37 °C showed complete digestion; proofreading-PCR to avoid A-Tailing (Phusion), ligation </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row49">
+		<td class="col0">11.08.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">amplification of T4MBP by adding EcoRI and NcoI sites with the primers 8 and 9; digestion of amplificat and pASK vector with EcoRI and NcoI; simulatnous dephosphorlyation of pASK, ligation of pASK and T4-MBP (16 h, 16 °C) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row50">
+		<td class="col0">10.08.2014 </td><td class="col1">Andi </td><td class="col2">IPG </td><td class="col3">immunostain.v2 </td><td class="col4">see 07.08; still no difference between control and samples; 9 min are enough for the final incubation of substrate buffer </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row51">
+		<td class="col0">09.08.2014 </td><td class="col1">Andi </td><td class="col2">IPG </td><td class="col3">colony-PCR, SDS-PAGE.v2 </td><td class="col4">with oligonucleotides 729, 734 (BCH) &amp; Ta = 48 °C (1:30 Min) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row52">
+		<td class="col0">08.08.2014 </td><td class="col1">Andi </td><td class="col2">IPG </td><td class="col3">plasmid-isolation, pASK-traformation.v2 </td><td class="col4">Isolation of pORE-E3 for future cloning and EMP_in_pMA_Colony1 for sequencing. Transformation of Kathis and Björns ligation-product in freshly prepared heatshock competent BL21 (DE3) pLyss cells. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row53">
+		<td class="col0">08.08.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">isolation of pASK from overnight culture </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row54">
+		<td class="col0">07.08.2014 </td><td class="col1">Andi </td><td class="col2">IPG </td><td class="col3">immunostain, colony-PCR, ONC </td><td class="col4">polyclonal anti-Flag-antibody were used as primary, and anti-rabbit alkaline phosphatase as secondary antibody (both 1:2000 diluted). after incubating the substrate buffer for 13 min, the same signals (all over the lanes; very unspecific) were observed in the samples and control, respectively. We were not sure, if problems in the sample handling are the cause for that, and suggest a repetition. Although colony-PCR (F1+R1) confirmed the insertion of GFP, another primer set (BCH: 1101+625) targeting the backbone of pMA did not bind in the colony, but showed a not expected amplificate length of ~1200 bp in the negative control (expected for pMA: ~1600 bp) ONC of pORE-E3, </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row55">
+		<td class="col0">07.08.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">only colonies on positive control → again inoculation of overnight culture </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row56">
+		<td class="col0">06.08.2014 </td><td class="col1">Anke, Andi </td><td class="col2">IPG </td><td class="col3">SDS-PAGE, Coomassie-stain &amp; Blotting </td><td class="col4">The volume of cellulose-bound protein samples were reduced by direct application of „Polyethylenglykol 6000“ on top of the tube. All  samples were loaded into a 12 % SDS-PAGE. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row57">
+		<td class="col0">06.08.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">Amplification of CDS (without HIS-TAG) with primers 8 and 9 (Botany) via Phusion. Purification of the product with „Wizard-PCR and Gel Kit) Double digest of PCR product and pASK vector with EcoRI and NcoI for 30 min. Purification of both samples. Poor results. Ligation of insert and pASK for 2 h (22 °C); transformation over night (37 °C) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row58">
+		<td class="col0">05.08.2014 </td><td class="col1">Andi </td><td class="col2">IPG </td><td class="col3">cellulose-bound-protein GFP_in_pMA_EMP2 </td><td class="col4">Dialyzing four times to remove Urea from the cellulose samples  (1x overnight, 2x during the day, 1x overnight). Repetion of EMP-PCR(2) reaction mix  no. 2 (protocol of Anke). After an agarosegel confirmed the correct fragment length, the reaction mix was purfied using the „Wizard-Kit“ (BCH), phosphorylated and ligated overnight according to Ankes protocol. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row59">
+		<td class="col0">05.08.2014 </td><td class="col1">Kathi, Björn </td><td class="col2">IPG </td><td class="col3">insertion of T4MBP in pASK </td><td class="col4">Isolation of Plasmid (pORE&#65279;_E3_2x35S_Expa_T4MBP_CBD) from E. coli overnight culture via Plasmidprep. MiniKit (Firma?). Amplification of CDS (without HIS-TAG) with Primers 8 and 9 (Botany) via Phusion. → did not work → again </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row60">
+		<td class="col0">04.08.2014 </td><td class="col1">Anke, Andi </td><td class="col2">IPG </td><td class="col3 leftalign">isolation of transient expressed protein from <em>N. tabacum </em>  </td><td class="col4">Isolation of cellulose-bound and unbound protein from N. tabaccum. The material was cooled by liquid nitrogen, automatically macerated by „Precellys“ and resuspended in 1 x SDS-sample buffer. Per plant one sample was taken. The debris-pellet from centrifugation was washed with ddH20 two times and incubated in 1 ml 8 M Urea overnight. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row61">
+		<td class="col0">31.07.2014 </td><td class="col1">Steffen, Fabian </td><td class="col2">Botany </td><td class="col3 leftalign">floral dip transformation of <em>A. thaliana</em>  </td><td class="col4">Transformation of A. thaliana via Floral Dip by using <em>A. tumefaciens</em> as vector. Construct: pORE&#65279;_E3_2x35S_Expa_T4MBP_CBD </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row62">
+		<td class="col0">31.07.2014 </td><td class="col1">Anke, Björn </td><td class="col2">IPG </td><td class="col3">transient transformation of <em>N. tabacum</em> with pORE&#65279;_E3_2x35S_Expa_TMBP_CBD </td><td class="col4">transient Transformation of 5 <em>N. tabacum</em> plants with pORE_E3_2x35S_TMBP in GV1301 and 4 plants without GV1301. Using 1ml / leave and two leaves / plant. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row63">
+		<td class="col0">25.07.2014 </td><td class="col1">Anke </td><td class="col2">IPG </td><td class="col3">colony-PCR of EMP2-transformation </td><td class="col4">Colony-PCR with primers 1011 and 625 (IPG) and 10 new colonies. Poor result… </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row64">
+		<td class="col0">23.07.2014 </td><td class="col1">Anke </td><td class="col2">IPG </td><td class="col3">colony-PCR of EMP2-transformation </td><td class="col4">Colony-PCR with primers 1011 and 625 (IPG). Poor result…. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row65">
+		<td class="col0">22.07.2014 </td><td class="col1">Anke </td><td class="col2">IPG </td><td class="col3">transformation of <em>E. coli </em> with EMP2 </td><td class="col4">Transformation of XL1-Blue Competent Cells </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row66">
+		<td class="col0">21.07.2014 </td><td class="col1">Anke,    <br>
+Andi </td><td class="col2">IPG </td><td class="col3">EMP 2 GfP </td><td class="col4">Screening the functional amount of megaprimer with and without F1: 20 ng Megaprimer + F1 + R2 </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row67">
+		<td class="col0">21.07.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">colony-PCR of Agor-Trafo </td><td class="col4">Colony-PCR with primer 11 (AG Offermann and 1484). Poor result, proabably of too high annealing temperature. New colony-PCR with 11 and 1261. Prepare 2 days cultures for Arabidopsis transformation </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row68">
+		<td class="col0">18.07.2014 </td><td class="col1">Anke </td><td class="col2">IPG </td><td class="col3">EMP 1 GfP </td><td class="col4">EMP1 with F1, R2 and GFP-vector to genererate Megaprimer. Result is fine. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row69">
+		<td class="col0">18.07.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">transformation of Agrobacterium with pORE&#65279;_E3_2x35S_Expa_TMBP_CBD </td><td class="col4">Using electrocompetent GV3101 cells. </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row70">
+		<td class="col0">17.07.2014 </td><td class="col1">Steffen, Fabian </td><td class="col2">Botany </td><td class="col3">plasmidpreparation and sequencing </td><td class="col4">Using ONC of <em>E. coli</em> with pORE and insert. Plasmidprep via Thermo Kit. Sequencing from both directions with primer 11 (AG Offermann) and 1261. Poor results for reverse direction. Resequencing of reverse sequence with 1484 </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row71">
+		<td class="col0">15.07.2014 </td><td class="col1">Steffen, Fabian </td><td class="col2">Botany </td><td class="col3">colony-PCR of pORE with Insert </td><td class="col4">Taq-PCR of 27 colonies using primer 11 of AG Offermann (binds to 35S) and 1012. Poor result, wrong reverse primer used. Doing new colony PCR with other reverse primer 1261 (binds to NOS-terminator. Result is fine. ONC </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row72">
+		<td class="col0">14.07.2014 </td><td class="col1">Steffen, Fabian </td><td class="col2">Botany </td><td class="col3">cloning of CDS (Expa_T4MBP_CBD) into pORE2x35S </td><td class="col4">1. PCR-Purification of Phusion-PCR (11.7) to get rid of disturbing enzymes. 2.) double digest of purified PCR-product and vector with MluI and BamHI for 1 h. 3.) Preparative Gelelctrophoresis. Cutting out specific bands. 4.) Gelextraction. 5.) Ligation of digested insert and vector for 1 h. 6.) Transformation of chemical competent E. coli DH5alpha with ligated DNA via heatshock </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row73">
+		<td class="col0">11.07.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">amplification of CDS from polyprotein </td><td class="col4">Phusion-PCR. Using Geneart-Shipping vector with insert as template. Primers 106 and 859 </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row74">
+		<td class="col0">03.07.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">negative colony-PCR </td><td class="col4">(continued by Fabian and Steffen) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row75">
+		<td class="col0">02.07.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">repetition of day 27.06.2014: Transformation of XL1blue with T4MBP in pORE-E3 with 35S-Promotor </td><td class="col4 leftalign">  </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row76">
+		<td class="col0">01.07.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">due to unsuccessful transformation repetition of day 26.06.2014: ligation of T4MBP with pORE-E3 with 35S-Promotor </td><td class="col4">Digestion of pASK with T4MBP and pORE-E3 with  2x35S promotor with MluI and BamHI, overnight ligation </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row77">
+		<td class="col0">30.06.2014 </td><td class="col1">Andi </td><td class="col2">IPG </td><td class="col3">sequencing Bielefelder-CBDs </td><td class="col4">Sequencing (Seqlab/ Microsynth) was done with primer no. 611 (GCTGGCCTTTTGCTCAGATGTTCTTTCCTGCGTTATC) and revealed that the obtained sequencing result  matched the given sequence online </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row78">
+		<td class="col0">30.06.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">negative colony-PCR </td><td class="col4 leftalign">  </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row79">
+		<td class="col0">27.06.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">transformation of XL1-Blue with T4MBP in pORE-E3 with 35S-Promotor </td><td class="col4 leftalign">  </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row80">
+		<td class="col0">26.06.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">ligation of T4MBP with pORE-E3 with 35S-Promotor </td><td class="col4">Digestion of pASK with T4MBP and pORE-E3 with  2x35S promotor with Mlu1 and BamHI, overnight ligation </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row81">
+		<td class="col0">26.06.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">plasmidpreparation of pASK </td><td class="col4">Digestion of pASK with T4MBP and pORE-E3 with  2x35S promotor with Mlu1 and BamHI, overnight ligation </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row82">
+		<td class="col0">25.06.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">colony-PCR of transformated XL1blue with T4MBP in pASK </td><td class="col4">Primer for colony PCR: T7 and BackSeq-pGII </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row83">
+		<td class="col0">24.06.2014 </td><td class="col1">Kathi </td><td class="col2">IPG </td><td class="col3">transformation to back up the synthesised T4MBP located in pASK vector </td><td class="col4">Transformation of XL1-Blue Competent Cells </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row84">
+		<td class="col0">24.06.2014 </td><td class="col1">Melanie </td><td class="col2">IPG </td><td class="col3">linearization of Bielefelder-CBDs </td><td class="col4"> <br>
+linearizing BBa_K863101 &amp; BBa_K863111 </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row85">
+		<td class="col0">24.06.2014 </td><td class="col1">Melanie, Andi </td><td class="col2">IPG </td><td class="col3">isolation of Bielelfelder-CBDs </td><td class="col4">Isolating BBa_K863101 &amp; BBa_K863111 with PeqGOLD Plasmid Miniprep Kit by PeqLab GmbH and selfmade solutions </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row86">
+		<td class="col0">20.06.2014 </td><td class="col1">Andi </td><td class="col2">IPG </td><td class="col3">ONC of Bielefelder-CBDs </td><td class="col4">Culturing XL1 containg pSB1C3 plasmid with parts BBa_K863101 &amp; BBa_K863111 in 10 ml LB medium and 10 µl Chloramphenicol </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row87">
+		<td class="col0">19.06.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">plasmidpreparation </td><td class="col4">Preparation of the 3 <em>E. coli</em> clones which were expected to have the right insert (using Thermo Mini-Prep Kit). Plasmids of one all 3 clones were send for sequencing, using Primer 1011 (IPG). Sequencing worked fine. All 3 clones have the insert at the right position </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row88">
+		<td class="col0">18.06.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">new colony-PCR </td><td class="col4">New Colony-PCR for detecting transformed E. coli clones. Using primers 1011 and 1012 (IPG). Detecting 3 positive clones wich were used for over-night cultures </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row89">
+		<td class="col0">16.06.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">plasmidpreparation </td><td class="col4">Preparation of 2<em>E. coli</em> clones wich were expected to have the right insert (using Thermo Mini-Prep Kit). Plasmid of one clone was send for sequencing, using Primer 962 (IPG). Sequencing didn't work (second sequncing trial by seqlab failed as well). <strong>Probably Primer 962 isn't working</strong> (even without 2x35S insertion sequencing should have work) </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row90">
+		<td class="col0">13.06.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">colony-PCR </td><td class="col4">Colony-PCR for detecting transformed <em>E. coli</em> clones. Using amplification primers 01 and 02 AND 1011 (IPG). Colony-PCR worked more or less, results were interpreted wrongly. False clones were used for over-night cultures </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row91">
+		<td class="col0">12.06.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">digest, ligation and transformation of 2x35S into pORE </td><td class="col4">1) Gelextraction of 2x35S (using Qiagen-Kit). 2) Digest of 2x35S and pORE with XhoI and BamHI (37 °C for 1 h). 3) Gelelectophoresis for separation of cutted pORE, using the large fragment (Gelextraction). 4) Ligation of 2x35S and pORE (using T7-Ligase). 5) Transformation of chemical competent E. coli DH5-Alpha and plating on LB-plates with Kanamycin </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row92">
+		<td class="col0">10.06.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">amplification of 2x35S Promotor </td><td class="col4">Using Primer 01 and 02 to amplify 2x35S promotor from template DNA. Separation of fragments via gelelectophoresis and preparation on fragments from the gel </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row93">
+		<td class="col0">21.05.2014 </td><td class="col1">Fabian </td><td class="col2">Botany </td><td class="col3">BamHI, MluI-digest </td><td class="col4">Analysing if both Enzymes (BamHI and MluI) cut while double digesting pORE E3 </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row94">
+		<td class="col0">19.05.2014 </td><td class="col1">Anke </td><td class="col2">IPG </td><td class="col3">- </td><td class="col4">Preparing Glystock </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row95">
+		<td class="col0">19.05.2014 </td><td class="col1">Steffen, Fabian </td><td class="col2">Botany </td><td class="col3">- </td><td class="col4">plasmid isolation pORE-E3 </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+	<tr class="row96">
+		<td class="col0">16.05.2014 </td><td class="col1">Steffen, Andi </td><td class="col2">IPG </td><td class="col3">„over-weekend“- culture pORE-E3 </td><td class="col4">Culturing JM109 bacteria cells containing pORE-E3 (250 µM from Glystock) in 10 ml LB media including 10 µM Kanamycin (Stock?) over weekend at 27 °C </td><td class="col5 leftalign">  </td><td class="col6 leftalign">  </td>
+	</tr>
+</tbody></table>
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