Lab Notebook - Elicia

Notebooks Home Page

JUNE

6-22-09

Followed Protocol( Revisions were made for a triple cell content) Each well had 200 uL of the cells Check OD Measurements
Strep Trial 1

Wells	ABC 9 (-) w/o arabinose	ABC 10(-) w arabinose	ABC 11(+) w/o arabinose	ABC 12(+) w/ arabinose
OD 1	0.4940	0.4092	0.4945	0.2558
OD 2	0.5201	0.4246	0.5281	0.2559
OD 3	0.4603	0.3665	0.4508	0.2010

File:Nowashtop.jpg
Last 4 Wells No Wash- top

File:Nowashbottom.jpg

Last 4 Wells-bottom Flipped upside down no wash
5uL of strep/PBS (1 part to a part was added to the wells
Incubation for 45 min
Cells were spinned down (followed protocol)

File:Somewashtop.jpg

(Last 4 Wells top Wells A 9-12 and B12 (washed only))
Cells Wells A 9-12 and B12 were washed with 100 uL PBS

Wells	9 (-) w/o arabinose	10(-) w arabinose	11(+) w/o arabinose	12(+) w/ arabinose
A	13	350	201	40
B	50	71	38	50
C	43	41	36	4059

File:Washtop.jpg
(Last four Wells-top Wash)
All Wells were washed with 100 uL PBS (Wells A 9-12 and B12 were not rewashed)

Analysis of Results

Not what we wanted to see
Well C-12 supernatant was still in the well before PBS was added?

What we will do….

Revise the strep protocol
Use a new positive control pBca 9145- Bca 9494

JUNE

6-23-09

diluted strep plasmids

JUNE

6-24-09

Bio277

Oligo	Foward	Reverse	DNA Template
328	E4	F4	Cellulose(red writing on tube)
362	C4	D4	E2348/69
324	A4	B4	MG1655
283	Ca998	G5	M10088
282	Ca998	F5	M10014
281	Ca998	E5	M10052
280	Ca998	D5	M10044
279	Ca998	C5	M10011
278	Ca998	B5	M10086
277	Ca998	A5	M10026

Analytical Gel

induced strep plasmids. Parts and vector(??) were digested

JUNE

6-25-09

Did ligation(vector used ??), transformation(used TG1 cells- plated on amp)and strep

JUNE

6-26-09

Redid ligation(used pBca 9523-1144 #5) and transformation (used TG1 cells plated on Spec and did heat shock)
....awaiting results (keeping fingers crossed)
....awaiting new streptavin to arrive on Monday June 29

JUNE

6-28-09

Plates are beautiful- no red and big white cells are present.
Annoculated (+) and (-) controls for Strep (oh no induced overnight and did a 2% concentration- that's not right always do a 1% and induce in the morning so it can be used in 3-6 hours- was redid by Tom)

JUNE

6-29-09

Mini Prep
(used zymo columns- must redo)

JUNE

6-30-09

Redid Mini Prep

Analytical Gel

Send parts for Sequencing yayyy...

July

7-3-09

Part	Eig	Sequence Result	Actual Sequence	Result
328	Eig53/54	File:Eig053.ab1 File:Eig054.ab1	File:Basicparts.txt	Perfect
326	Eig 41	File:Eig041.ab1	File:Basicparts.txt	Perfect
324	Eig 56	File:Eig56.ab1	File:Basicparts.txt	Perfect
283	Eig 51/52	File:Eig051.ab1 File:Eig052.ab1	File:Basicparts.txt	Missence Mutation
283	Sig 86	G5	File:Basicparts.txt	Missence Mutation
282	Eig 42	File:Eig042.ab1	File:Basicparts.txt	Perfect
282	Sig 85	F5	File:Basicparts.txt	Perfect
281	Eig 47/48	File:Eig047.ab1 File:Eig048.ab1	File:Basicparts.txt	Perfect
280	Eig 49/50	File:Eig049.ab1 File:Eig050.ab1	File:Basicparts.txt	Perfect
279	Eig 45/46	File:Eig045.ab1 File:Eig046.ab1	File:Basicparts.txt	Perfect
278	Eig 43/44	File:Eig043.ab1 File:Eig044.ab1	File:Basicparts.txt	Perfect
277	Eig 55	File:Eig55.ab1	File:Basicparts.txt	Perfect

Reaequenced Bioig 277 and 324

Sent for Sequencing Eig55 and 56

July

7-6-09

bowling

July

7-8-09

Poured spec and amp plates. Autoclaved glassware. Set up strepspread sheet.

July

7-24-09

K11212 that was sent off for sequencing

July

7-25-09

Mini prep 24- well plate samples. Only Bdr0ll-2(Aa),Bdr0ll-1 #2(Ba),MV45-2(Ca), Bdr0ll-2#2(Da), Bdr0ll-1#3(Ea),Bdr0ll-2 #3(Fa),Bdr0ll-1#1(Ga)

Order is:Bdr0ll-2(Aa),Bdr0ll-1 #2(Ba),MV45-2(Ca), Bdr0ll-2#2(Da), Bdr0ll-1#3(Ea),Bdr0ll-2 #3(Fa),Bdr0ll-1#1(Ga) Colony PCR from Plates
MV45-1(Ab), 009-1(Bb),k11212-2(Cb), 008-1(Db),009-2(Eb), MV45-2(Fb), k112126-1(Gb), k112126-2(Hb), k11212-1(Ib),008-2(Jb)
Order is:MV45-1(Ab), MV45-1(Ab),009-1(Bb),009-1(Bb),k11212-2(Cb),k11212-2(Cb), 008-1(Db),Nothing, Marker, 009-2(Eb), 009-2(Eb),MV45-2(Fb), MV45-2(Fb),k112126-1(Gb),k112126-1(Gb), k112126-2(Hb), k112126-2(Hb),k11212-1(Ib),k11212-1(Ib),008-2(Jb),008-2(Jb)

July

7-26-09

Redid PCR
k112126-1(Ac),k112126-1(Bc),008-1(Cc),008-1(Dc),009-1(Ec),-009-2(Fc),MV45-1(Gc),MV45-2(Hc),k11212-1(Ic),k11212-2(Jc)

July

7-27-09

k112126-1(Ac),k112126-1(Ac),k112126-1(Bc),k112126-1(Bc),008-1(Dc),008-1(Dc),009-1(Ec),009-1(Ec),009-2(Fc),009-2(Fc),MV45-1(Gc),MV45-1(Gc),MV45-2(Hc),MV45-2(Hc),k11212-1(Ic),k11212-1(Ic),k11212-2(Jc)k11212-1(Ic),(008-1 Cc turned pink- so it was not included in this gel) Grew in LB AMp:k112126-1(Ac),k112126-1(Bc),008-1(Cc),008-1(Dc),009-1(Ec),-009-2(Fc),MV45-1(Gc),MV45-2(Hc),k11212-1(Ic),k11212-2(Jc)

July

7-28-09

Miniprep Fig3 parts only k112126-1(Ad),k112126-1(Bd),008-1(Dd),MV45-2(Hd),k11212-1(Id),k11212-2(Jd) Gc,Ec: did not grow
Fc:turned pink File:Fig4ecf.jpg

.