Lab Notebook - Elicia
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
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)
(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
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 |
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 | Eig53/54 | 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
.