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From 2014.igem.org

Notebook / Plant Vector

Here we list all the steps that were tested to modify the pORE E3 (AY562536.1) into a vector with BioBrick RFC[21]MCS and 2x35S promotor.

ONC = overnight culture ; CBD = Cellulose binding domain ; IPG = Institute for plant genetics ; OD = optical density ; PCR = Polymerase chain reaction ; RT = room temperature ; T4MBP = Top 4 metal binding protein ; RFC[21] = assembly standard 21 ; TR = Thomas Reinard

date	coworkers	lab	activity	short summary
25-Sept-2014	Lisa	IPG	colony-PCR	colony-PCR with primer 1261 and 488: all 24 colonies negative or inconclusive/ no more time to do the insertion of rcf again
24-Sept-2014	Steffen, Lisa	IPG	triple digest/ oligo annealing	vector: triple digest: gel extraction/ insert: oligo annealing/ phosphorylation/ ligation of vector and insert/ transformation of XL1-Blue Competent Cells/ incubation over night (25 °C)/ everything performed after modified protocol
23-Sept-2014	Steffen	IPG	plasmidpreparation/ amplification of pORE_E3	plasmidpreparation of ONC from Katharina/ amplification
22-Sept-2014	Lisa, Katharina	IPG	insertion of RFC[21]	colony-PCR: only negative controls/ consultation of TR: modification of the protocol
19-Sept-2014	Katharina	IPG	insertion of RFC[21]	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)
18-Sept-2014	Katharina	IPG	insertion of RFC[21]	digestion of pORE-E3 (without XhoI- and BglII-site): no DNA: inoculation of an ONC
17-Sept-2014	Katharina, Björn	IPG	insertion of RFC[21]	colony-PCR with primer 1261 and 488: all 60 colonies negative: digestion probably went wrong/ isolation of plasmid of another ONC via handpreparation
16-Sept-2014	Katharina, Björn	IPG	insertion of RFC[21]	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
15-Sept-2014	Katharina, Björn	IPG	insertion of RFC[21]	digestion of pORE-E3 (without XhoI and BglII-site): no DNA: inoculation of an ONC
27-Aug-2014	Melanie, Lisa	IPG	plasmidpreparation/ sequencing.v1	plasmidpreparation with 12 colonies/ double digestion (XhoI and NcoI): 7 might be without BglII AND XhoI: sequencing with primer ??? (IPG) positive/ pORE_E3 2x35S (without T4MPB) and without BglII- and XhoI-site
26-Aug-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-Aug-2014	Melanie, Lisa	IPG		ligation/ transformation of XL1-Blue Competent Cells via heat-shock/ selection on kanamycin
22-Aug-2014	Melanie	IPG	digest of pORE_E3 2x35S (without T4MBP and without BglII-site) with XhoI, proofreading PCR	digestion of pORE_E3 2x35S (without T4MBP and without BglII-site) with XhoI for 1h 37 °C showed complete digestion/ proofreading-PCR to avoid A-Tailing (Phusion)
20-Aug-2014	Melanie	IPG	sequencing.v3 (new primer)	sequencing with primer 1356: positive: BglII-site destroyed
19-Aug-2014	Melanie	IPG	sequencing.v2 (same primer)	sequencing with primer 1261 (IPG) again stopped 10 bp before BglII-site: no definite result: again
15-Aug-2014	Lisa	IPG	plasmidpreparation/ sequencing.v1	plasmidpreparation with 10 colonies/ digestion with BglI (twice): poor results: 1 probe might be without BglII: sequencing with primer 1261 (IPG) of this probe stopped 10 bp before BglII-site: no definite result: again
13-Aug-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-Aug-2014	Melanie	IPG	transformation of E. coli with pORE_E3 2x35S (without T4MBP) ?without? BglII	transformation via heat-shock/ selection on kanamycin
11-Aug-2014	Melanie, Lisa	IPG	digest of pORE_E3 2x35S (without T4MBP) with BglII/ proofreading PCR/ ligation	digestion of pORE_E3 2x35S (without T4MPB) with BglII for 1h 37 °C: complete digestion/ proofreading-PCR to avoid A-Tailing (Phusion)/ ligation