Lab Notebook - Elicia

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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 0.49400.4092 0.49450.2558
OD 20.52010.4246 0.52810.2559
OD 3 0.46030.3665 0.45080.2010

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

Image: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)

Image: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 13350 20140
B5071 3850
C 4341 364059


Image: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 E4F4 Cellulose(red writing on tube)
362C4D4 E2348/69
324 A4B4 MG1655
283 Ca998G5 M10088
282Ca998F5 M10014
281 Ca998E5 M10052
280Ca998D5 M10044
279 Ca998C5 M10011
278 Ca998B5 M10086
277 Ca998A5 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

7-1-09

7-2-09

7-3-09

Part Eig

Sequence Result

Actual Sequence

Result

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



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 Image:Fig4ecf.jpg










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