ApoControl Notebook

Contents

8-02-2010

• Some test text here.

8-03-2010

Some test text in bold We created following tests:

• test1
• test2
• test3

- test4
- test5

8-04-2010

Example of a table

8-05-2010

this is also a table:

table with 3 cells

8-06-2010

text

8-07-2010

text

8-08-2010

test
test

8-09-2010

text Knallroter Text
farbnummern für farbige schrift: http://html.nicole-wellinger.ch/hilfen/farbenverzeichnis.html

test grüner text

8-10-2010

Transforming competent cells

- eGFP Biobrick: BBa_I714891 SDY_eGFP (Kanamycin)

- TEV recogn N Degron SF3 = pDS7 (Ampicillin)

- TEV p14 recogn = 190-6 (Ampicillin)

-> Protocol: (3 Transformation)

- We added 2 µl DNA

- We plated out 200 µl

Plasmid Isolation

- CMV-Promoter Biobrick: BBa_J52034

-> Protocol:(4 Plasmid extraction from cells)

- Prepared overnight culture, measured concentration of DNA

-> Poor results -> thrown away

8-11-2010

New Plasmid Extraction

- CMV-Promoter Biobrick: BBa_J52034

-> Protocol: (4 Plasmid extraction from cells)

- Plasmid concentration: 143ng/µl

Prepared overnight culture of eGFP BBa_I714891

- 3 ml LB-Media + 4 µl Kanamycin

- Inoculated iangeimpft) with 1 colony of BBa_I714891 -> 37°C

Prepared overnight culture of 190-6 and pDS7 and eGFP (BBa_I714891) in falcons

- for 190-6 and pDS7: 10µl Ampicillin + 10 ml LB-Media + colony of plate

- for eGFP: 13,3 µl Kanamycin + 10 ml LB-Media + 1 colony of plate

Restriction digestion of CMV-Promoter BBa_J52034 with EcoRI and PstI

-> mixed

- plus: EcoRI (10µg/µl): 0,5 µl resp. PstI (10µg/µl): 0,5 µl

- incubated at room temperature from 12:10 to 15:00, 1 hour at 37°C, 2 hours at 60°C

- frozen at -20°C

Prepare new/fresh overnight culture of CMV-Promoter Biobrick: BBa_J52034

- 1 ml of "old" culture + 3 ml LB-Media + 4 µl Kanamycin -> 37°C

8-12-2010

Plasmid Extraction of pDS7, eGFP, 190-6

-> Protocol: (4 Plasmid extraktion from cells)

- pDS7 (458ng/µl), eGFP (55ng/µl), 190-6 (193ng/µl)

Restriction digest of pDS7, eGFP, 190-6

- with EcoRI and PstI in buffer H (for testing DNA is correct)

-> Protocol: (5 Restriction digest)

- 10µg DNA: pDS7 (2µl), eGFP (15µl), 190-6 (10µl)

Plate colonies for plasmid extraction

- CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin))

- PhiC31o plated on Ampicillin-Agar, stored at 37°C

50% Glycerol made

- for PhiC31o glycerol stock (produced later)

8-13-2010

Gelfoto from the EcoR1 and Pst1 Restrictiondigest of 190-6, eGFP, pDS7 and CMV

Inoculate CMV into LB medium with ampicillin

- CMV (BBa_J52034) from 10.8.2010 inoculated into LB medium with ampicillin, as falsly inoculated in Kanamycin

Agarosegelelectrophoresis with digestions

->Protocol (11 Agarose gel electrophoresis)

- Agarosegelelectrophoresis with the digestions (CMV, eGFP, pDS7, 190-6), 125V for 30 minutes and then for 20 minutes;

- expected DNA bands: 190-6 (4840bp, 1903bp), pDS7 (8027bp, 6bp), CMV (654 bp (Insert), 2079bp (Plasmid)), eGFP (720bp (Insert), 2750bp (Plasmid))

- Correct DNA bands for 190-6 (~4800bp, ~1900bp, ~6700bp (undigested plasmid)) and eGFP (~2000bp (Plasmid), ~750 bp (Insert)); CMV probably not digested (two bands; one probably normal, one supercoiled) and pDS7 not clear

Restriction digest from CMV and pDS7

-> Protocol (5 Restriction digest)

- Restriction digest from CMV (EcoR1, Pst1; 6µl DNA, buffer H) and pDS7 (EcoR1, Spe1; 2µl DNA, buffer B)

Agarosegelectrophoresis with digestions

->Protocol (11 Agarose gel electrophoresis)

- Agarosegelelectorphoresis for 30 minutes, 150V

- Expected DNA bands: CMV see above, pDS7 (3647bp, 3369bp, 1011bp, 6bp)

- false DNA bands CMV (~1200 bp, ~2000 bp) and pDS7 (~8000bp two bands, ~1100 bp); required to isolate a new colony for these two Plasmidextractions

Plated CMV on Ampicllin-Agar

- Plated the colony from CMV (BBa_J52034) for Plasmidextraction (Ampicillin), as falsly plated on Kanamycin

8-14-2010

weekend

8-15-2010

weekend

8-16-2010

Planting colonies

- transfer 1 ml PhiC31o culture to new LB medium + Amp, 37°C

- pick up CMV and pDS7 colonies from plates and transfer to LB medium+Amp, 37°C

Plasmid Extraction of PhiC31o

->Protocol (4 Plasmid extraktion from cells)

- plasmid extraction of PhiC310

->27,5ng/µl DNA and second plasmid extraction of PhiC310 (i. o. to get more DNA); first eluation-step with first eluation-extraction

-> 60ng/µl DNA

Restriction digest

->Protocol (5 Restriction digest)

- restriction digest of PhiC310 with EcoR1 and Spe1

restriction digest in the thermo cycler (program "Verdau", see protocol)

Handling primers after arrival (1,2,3,4,5,6,11,12)

->Protocol (9 Handling primers)

PCR preparations

- 10mM dNTP mix made from 100 mM dATP, dGTP, dCTP, dTTP by taking 100µl of each and adding 600µl H ₂ O

PCR 1 and 6

- PCR of the tet inducible CMV minimal promotor out of prevTRE (=PCR 1 with Primer 1 and 2) and SV40PA out of pcDNA3 (=PCR 6 with Primer 11 and 12)

->Protocol (10 PCR with Pfu)

Mixture:

Programme:

Glycerolstock of PhiC31o

- Glycerolstock of the colony of PhiC31o for the plasmidextraction

8-17-2010

Agarose gel electrophoresis of PCR6 which shows that PCR6 is about 200bp

Plate CMV and pDS7 colonies on Ampicillin-Agar

- colonies for plasmidextraction of CMV and pDS7 plated on Ampicillinplates

Plasmid Extraction of CMV and pDS7

- plasmidextraction of CMV (2,5ng/µl) and pDS7 (10ng/µl) the A260/A280 value was 1.333, which means that it was 90% Protein and only 10% DNA (should be 1,8); new plasmidextraction needed

new overnight cultures of CMV and pDS7 for a new plasmidextraction made

Agarose gel electrophoresis

-> Protocol (11 Agarose gel electrophoresis)

- Agarose gel electrophoresis of the restriction digest of PhiC31o and PCR 1 and 6

- the right bands found for PhiC31o (~2900,~2400,~250)

- the right band found for PCR1 (~450)

- no band found for PCR6; new electrophoresis needed with more DNA loaded

Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6	Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6 which shows that PCR1 is between 250 and 500 bp

- new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown)

- the right band found for PCR6 (~200)

New overnight cultures of CMV and pDS7

- the overnight colonies didn't grow; new colonies (CMV and pDS7) picked from plate and inoculated in LB Ampicillin

PCR purification of PCR 1 and 6

-> Protocol (12 Gel extraction or PCR Clean up)

- DNA concentration of the PCR 1 and 6 products measured: PCR1: 410ng/µl (A260/A280=1.253) PCR6: 568ng/µl (A260/A280=1.275)

- PCR Purification with Promega Kit

-> PCR1: 230ng/µl (A260/A280=1.769)

-> PCR6: 37.5ng/µl (A260/A280=1.667)

8-18-2010

Agarose gel electrophoresis of (from left to right) CMV and pDS7 showing the right bands for pDS7

Plasmid Extraction of CMV and pDS7

-> Protocol (4 Plasmid extraction from cells)

- Plasmid extraction of CMV (97.5ng/µl; A260/A280=1.857) and pDS7 (212ng/µl; A260/A280=1.848)

Restriction digestion

-> Protocol (5 Restriction digestion)

- Restriction digestion of CMV (EcoR1 + Pst1; 10µl DNA, buffer H) and pDS7 (EcoR1 + Spe1; 5µl DNA, buffer B)

-> expected DNA bands: CMV: 2079bp (plasmid) + 654bp (Insert); pDS7: 7022bp + 1011bp

Agarose Gel electrophoresis of digested CMV and pDS7

-> Protocol (11 Agarorse gel electrophoresis)

-> right DNA bands for pDS7 (~7000bp, ~1000bp)

-> false DNA bands for CMV

- Starting PCR 2a and 2b (replication and mutagenesis of pDS7): 3 µl DNA and 50°C Annealing Temperatur (other same as 8-16-2010)

8-19-2010

Agarose gel electrophoresis of PCR 2a and 2b

-> Protocol (11 Agarose gel electrophoresis) (150V, 30min)

Agarose gel electrophoresis of (from left to right) PCR2a and PCR2b

-> the right bands for PCR2a (~300bp) and PCR2b (~700bp)

- New agarose gel electrophoresis with all of the PCR product for gel extraction (150V, 30min)

Gel extraction of the DNA from PCR2a and PCR2b

-> Protocol (12 Gel extraction or PCR Clean up)

- DNA concentration measured; problem with nanodrop as too low concentration; lyophille used to reduce volume

- DNA concentration measured again: PCR2a: 70ng/µl A260/A280=1.647; PCR2b: 45ng/µl A260/A280=1.5

PCR 3 (joining PCR of 2a and 2b)

- PCR3 (the joining PCR of PCR2a and 2b; Joining of the TEVrecogn-N-Degron-SF3 part) done: 1.3 µl of PCR2a and 4.7 µl of PCR2b makes 300ng of a 1:1 solution of both to be joined DNA parts. Annealing temperature: 50°C

-> Protocol (10 PCR with Pfu)

8-20-2010

Agarose gel electrophoresis of PCR3

left column: marker; most right column: PCR3

-> Protocol: 11 Agarose gel electrophoresis (150V, 30min)

-> expected band: ~1000bp

-> false band: ~500bp

- probable reason: mini photometre was influenced by gel extraction chemicals, therefore it measured false DNA concentrations and false template masses were calculated

-> New 2a and 2b PCR

New PCR (2a and 2b)

-> Protocol: 10 PCR with Pfu

(see 8-18-2010, but 35,5µl water)

8-21-2010

weekend

8-22-2010

weekend

8-23-2010

Agarose gel electrophoresis of PCR 2b

-> Protocol: 11 Agarose gel electrophoresis

- expected band: 700bp

-> no band shown on gel -> new PCR 2b

PCR 2b

- start PCR 2b with PCR 2b from 8-13-10 as template ( 1:20 and 1:100 diluted; 1µl)

-> Protocol: 10 PCR with Pfu

- annealing temperature: 50°C; amount of water: 37,5µl

Agarose gel electrophoresis of PCR 2b 1:20 and 1:100

-> Protocol: 11 Agarose gel electrophoresis

- expected bands: each ~ 700bp

- false bands: ~ 200bp

-> new PCR with 2ng, 5ng, 10ng template pDS7

PCR 2b with 2ng, 5ng, 10ng template pDS7

- pDS7 1:100 diluted(-> 2,1 ng/µl)

Mixture:

-> Protocol: 10 PCR with Pfu

PCR 2a gel extraction

- Quaigen kit (QuaiexII)

-> Protocol: 14 QIAEX II gel extraction

Start 3 CMV overnight cultures

8-24-2010

agarose gel electrophoresis of PCR 2b

-> Protocol: 11 Agarose gel electrophoresis

Agarose gel electrophoresis of (from left to right) PCR2b (2ng (cut out), 10ng, 5ng template) showing the right bands for 2ng, 5ng template

- expected bands: right bands with 2ng and 5ng template (~700bp), no band with 10ng template

CMV plasmid extraction

-> Protocol: 4 Plasmid extraction from cells

Plasmid extractionof 3 different overnight cultures.

- results:

• 52,5 ng/µl A260/A280= 1.312
• 133 ng/µl A260/A280= 1.710
• 80 ng/µl A260/A280= 1.600

CMV restriction digestion

-> Protocol: 5 Restriction digestion

- CMV restriction digest: EcoRI, PstI, buffer H

• 19µl, H₂O : 0µl
• 6µl, H₂O : 9.5µl
• 12.5µl, H₂O : 3µl

PCR 2b gel extraction

- PCR2b was gel extracted (with Qiagen gel extraction kit), 17.5 ng/µl a260/A280= 1.750

-> Protocol: 14 QIAEX II gel extraction

PCR 3 (fusion of 2a and 2b)

- PCR3: conducted again at 52°C annealing temperature

• 10.5 ng (from PCR2b) 0.6µl

• 4.5 ng (from PCR2a) 0.9µl (1:10 diluted)

PCR2a	0.9 µl
PCR2b	0.6 µl
primer3	2.5 µl
primer6	2.5 µl
dNTPs	1 µl
Pfu	0.5 µl
10xbuffer	5 µl
H2O	37 µl

-> Protocol: 10 PCR with Pfu

agarose gel electrophoresis of CMV digestion

- agarose gel electrophoresis (150V, 25 min) of the CMV digestion

-> bands are wrong again ( ~ 1200bp, 2000bp)

8-25-2010

Agarose gel electrophorese of PCR 3

-> Protocol: 11 Agarose gel electrophoresis

- expected band: ~1000bp

- false band: ~400bp

Plasmid extraction of ccdB tet and ccdB strep

Plasmid extraction of pSB1C3 with BBa_P1010

-> Protocol: 4 Plasmid extraction from cells

- results:

Plate ccdB amp, cam, tet

- plate ccdB with ampicilline, chloramphenicol, tetracycline resistance on LB agar with appropiate antibiotic.

Overnight culture of ccdB kan

- Overnight culture of ccdB with kanamycine resistance in LB medium with kanamycine

PCR 7a, 7b, 9, 10

->Protocol: 10 PCR with Pfu

Mixture

Standard PCR; annealing temperature: 60°C

8-26-2010

Agarose gelelectrophoresis of PCR 7a, 7b, 9, 10

->Protocol: 11 Agarose gel electrophoresis

- 150V, 25min

Plasmid extraction of ccdB kan

-> Protocol: 4 Plasmid extraction from cells

-result: concentration: 25ng/µl; A260/A280= 2,0

New PCR 7a, 7b, 9, 10

Mixture

Program:

gradient PCR, 42-69°C annealing temp.

->Protocol: 10 PCR with Pfu

Overnight culture of ccdB amp, tet, cam

Inoculate one colony each in 5ml medium with approptraite antibiotic.

8-27-2010

Agarose gel electrophoresis of PCR 7a, 7b, 9, 10

->Protocol: 11 Agarose gel electrophoresis

150V, 25min, 75mA

from left to right: 7a, 7b, 9, 10, Marker

Plasmid extraktion of ccdB amp, tet, cam

->Protocol: 4 Plasmid extraction from cells

results:

New PCR 7a, 9

Mixture:

- 2ng template: see 26-8-10

- 4ng template: see 26-8-10, but 2µl template and 35,25µl water

-> Protocol: 10 PCR with Pfu

Restriction digestion of ccdB amp, kan, cam, tet

-> Protocol: 5 Restriction digestion

- only 90min 37°C incubation

- EcoRI, PstI, Buffer H

Agarose gelelectrophoresis of PCR 7a, 9, ccdB restriction digestion

150v, 25min, 75mA

-> Protocol: 11 Agarose gel electrophoresis

results:

- PCR7a, 9: false band at 200bp

- ccdB: each digestion leads to a right band with ~ 650bp

8-28-2010

weekend

8-29-2010

weekend

8-30-2010

New PCR 7a and 9

Mixture

-> Protocol: 10 PCR with Pfu

PCR program

PCR 7: Annealing Temperature 60°C - 25 x 1 min Annealing time and 5x 1,30 min Annealing time

PCR 9: Annealing Temperature 55°C - 25 x 1 min Annealing time and 5x 1,30 min Annealing time

Gel extraction of PCR 7b, 10

->Protocol: 14 QIAEX II gel extraction

results:

Agarose gel electrophoresis of new PCR 7a, 9

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

- results:

- 7a: no band shown

- 9: false band (~200bp)

New PCR 3

-> Protocol: 10 PCR with Pfu

PCR2a	0.9 µl
PCR2b	0.6 µl
dNTPs	1 µl
Pfu	0.5 µl
10xbuffer	5 µl
DMSO	1,25µl
H2O	36,75 µl
sum	45µl

- new method: standard PCR without primers (10 cycles, 56°C annealing temp.)

- then add 2,5µl of primer 3 and 6

- 30 cycles standard PCR (54°C annealing temp.)

8-31-2010

Gel photo of (left to right) PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu), PCR3(Phusion)

Agarose gel electrophoresis of PCR 4a, PCR4b, PCR3(Pfu), PCR3(Phusion)

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

- results:

- PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu): no band shown

- PCR3 (Phusion): right band (~1000bp)

New PCR PCR4a, PCR4b, PCR7a, PCR9

-> Protocol: 10 PCR with Pfu

PCR mixture for PCR4a, PCR4b

template	37.25 µl (200ng)
dNTPs	1 µl
Pfu	0.5 µl
10xbuffer	5 µl
DMSO	1,25µl
sum	50µl

Standard PCR program with annealing temperature PCR4a: 51.1°C, PCR4b: 48.5°C.

-> Protocol: 15 PCR with Phusion

PCR mixture for PCR7a, PCR9

template	4 µl (8ng)
dNTPs	1 µl
Phusion	0.5 µl
5xbuffer	10 µl
DMSO	1,25µl
H2O	28.25 µl
sum	50µl

PCR program: Phu62

98°C	1 min
98°C	10 sec
62°C	20 sec
73°C	30 sec
return to step 2 for 29 cycles
73°C	10 min
12°C	forever

Gel photo of (left to right) PCR3, ladder, PCR7a, empty, PCR9

Agarose gel electrophoresis of PCR 3, PCR7a, PCR9 for gel extraction

-> Protocol: 11 Agarose gel electrophoresis

120V, 30min

- results:

- PCR3; right band (~1000bp) and side-product

- PCR7a: no band

- PCR9: right band (~800bp) and side-product

Gel extraction of the DNA from PCR3 and PCR9

-> Protocol (12 Gel extraction or PCR Clean up)

results:

PCR 9: 22,5ng/µl; A260/A280=1,8

PCR 3: 22,5ng/µl; A260/A280=2,25

New PCR 4a, 4b, 7a with DreamTaq

-> Protocol: 16 PCR with DreamTaq

PCR mixture for PCR7a

template	5 µl (10ng)
dNTPs	5 µl
DreamTaq	0.33 µl
10xbuffer	5 µl
DMSO	1,25µl
H2O	28.5 µl
sum	50µl

Primers for PCR 7a: 13,14

Annealing temp: 60°C

PCR mixture for PCR4a,4b

template	36,5 µl (180ng HeLa cDNA)
dNTPs	5 µl
DreamTaq	0.33 µl
10xbuffer	5 µl
DMSO	1,25µl
sum	50µl

PCR 4a

Primers for PCR 4a: 7,8

Primers for PCR 4b: 9,10

Annealing temp: 50°C

PCR program:

95°C	1 min
95°C	30 sec
50/60°C	30 sec
72°C	1 min (1kb/min)
return to step 2 for 29 cycles
72°C	10 min
12°C	forever

9-01-2010

Agarose gel electrophoresis of PCR4a, PCR4b, PCR7 (DreamTaq)

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

results: no product

New PCR 4a, 4b with DreamTaq, Pfu, with concentration gradient and touch-down PCR

-> Protocol: 16 PCR with DreamTaq; 10 PCR with Pfu

PCR mixture for DreamTaq

Concentration	Low	Middle	High
template	5 µl (1:1000)	31.75µl (1:1000)	2µl (1:10)
dNTPs		5 µl
DreamTaq		0.33 µl
10xbuffer		5 µl
DMSO		1,25µl
H2O	26.75 µl	0	29.75
sum		50µl

PCR mixture for Pfu

Concentration	Low	Middle	High
template	5 µl (1:1000)	37.25µl (1:1000)	2µl (1:10)
dNTPs		1 µl
Pfu		0.5 µl
10xbuffer		5 µl
DMSO		1,25µl
H2O	32.25 µl	0	35.25 µl
sum		50µl

Primers for PCR 4a: 7,8; PCR 4b: 9,10

-> Protocol: Thermal cycler program: Touch down

Agarose gel electrophoresis of PCR4a, PCR4b

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

from left to right: 4a: P1, P2, P3, D1, D2, D3; 4b: P1, P2, P3, D1, D2, D3

key:

"P"= PCR with Pfu

"D"= PCR with DreamTaq

"1"= low template concentration

"2"= middle template concentration

"3"= high template concentration

expected bands:

• 4a: 330bp -> P2 and P3 show right bands and "primer clouds"(?)

• 4b: 376bp -> P1, P2, P3 show right bands and "primer clouds" (?)

9-02-2010

Agarose gel electrophorese of PCR 4a P2, 4b P2

-> Protocol: 11 Agarose gel electrophoresis

- 120V, 45min, 1,5% Agarose gel

Agarose gel electrophoresis of (from left to right) PCR4aP2, Marker and PCR4bP2

- Cut out bands at ~~ 350bp and extract

PCR Agarose gel extraction

-> Protocol: 14 QIAEX II gel extraction

results:

New PCR 7a with Pfu and Phusion

template: 190-6, Primer 13,14

Mixture with Pfu

-> Protocol: 10 PCR with Pfu

PCR mixture with Phusion

template (~2ng)	1,0 µl
dNTPs	1 µl
Phusion	0.5 µl
5xbuffer	10 µl
DMSO	1,25µl
H2O	31,25µl
sum	50µl

-> Protocol: 15 PCR with Phusion

New PCR 4a, 4b with Pfu

-> Protocol: 10 PCR with Pfu

-> Protocol: Thermal cycler program: Touch down

Mixture see 9-1-10, twice 4a and 4b

Agarose gel electrophorese of PCR 4a, 4b, 7a, gel extracted 4a and 4b

-> Protocol: 11 Agarose gel electrophoresis

- 25min, 150V

from left to right: 4a*, 4a, 4b*, 4b, 7a Phusion, 7a Pfu, Ladder

-> result: 4b, 4b*: right bands (~330bp)

-remain: false bands/no band

from left to right: ladder, 4 columns pathway, 4a gelextr., 4b gelextr.

-> results: slight right bands for 4a and 4b, no "primer clouds" anymore.

9-03-2010

Overlapping PCR 5 with Pfu and Phusion

template: 4a, 4b Primer 7,10

Mixture with Pfu

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Annealing temperature: 54°C

PCR mixture with Phusion

PCR4a (~15ng)	1.2µl
PCR4b (~15ng)	2µl (1:10)
dNTPs	1 µl
Phusion	0.5 µl
5xbuffer	10 µl
DMSO	1,25µl
H2O	29.05µl
sum	50µl

-> Protocol: 15 PCR with Phusion

PCR program: standard PCR program for Phusion, Annealing temperature: 58°C

New PCR7a with Pfu

template: 190-6; Primer: 13,14

Mixture with Pfu

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Gradient: 54.4°C, 57.8°C, 61.4°C, 65.0°C

-> results:

-7a: only "primer cluods"

-5 Pfu: no defined product (slurred?)

-5 Phusion: "primer clouds"

9-04-2010

weekend

9-05-2010

weekend

9-06-2010

charges for sequencing

every charge 7µl

New overlapping PCR 5 with Pfu

template: 4a, 4b; 15ng

Primer 7,10

Mixture

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Annealing temperature: 46.1°C,48.5°C, 51.1°C

-> results: "primer clouds"

New PCR 7a with Pfu new dilution of 190-6; 1:100

Agarose gel electrophoresis of 7a

-> results: "primer clouds"

charges for sequencing for PCR 1,7b and 9

every charge 7µl

9-07-2010

Agarose gel electrophorese of PCR gel extractions

150V, 25min

-> Protocol: 11 Agarose gel electrophoresis

-result:

from left to right: PCR 1,2a,2b,3,4a,/,7b,9,10,/,ladder,pathway

new PCR for PCR6

PCR mixture for PCR6

Concentration	Low	High
template	1 µl (1:100)	15µl (1:100)
primer (11,12)	2.5 µl*2
dNTPs	1 µl
Pfu	0.5 µl
10xbuffer	5 µl
DMSO	1,25µl
H2O	36.25 µl	22.25 µl
sum	50µl

-> Protocol: Thermal cycler program: Touch down, 62°C-52°C, 30 cycles by 55°C

9-08-2010

Restriction digestion of eGFP, PCR6, ccdBamp

Gel photo of (left to right) PCR6(5ng/100ng) template

as prepatation for ligation

-> Protocol 5 Restriction digestion

1:30h 37°C, 20min 80°C

Agarose gel electrophoresis of PCR6

-> Protocol: 11 Agarose gel electrophoresis

25min, 150V

Ligation 1

Ligation at 22,5°C for 30 min, denaturation at 65°C for 10 min.

Concentration of DNA in Ligation 1:

Transformation

-> Protocol 18 competent cells2

The incubation time for the cells is here 1 hour.

9-09-2010

Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9 Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9

-> protocol 11 Agarose gel electrophoresis

results: "primer-clouds", PCR 9: no band

Plasmid extraction of ccdBcam, ccdBamp, Bak, CMV, PhiC31o

-> Protocol 4 Plasmid extraction from cells

Eluated with H₂O instead of the Eluation Buffer.

New PCR 1, 3, 4a, 5, 7a, 9, 10 with phusion

+ in each assay:

dNTP mix: 1µl, 5x Phusion buffer: 10µl, DMSO:1.5µl, Phusion:0.5µl

sum: 50µl

Program: standard Phusion PCR, 29 cycles; annealing temperatures: see above

-> Protocol: 15 PCR with Phusion

[from left to right: PCR 1, 3, 4a, 5, 7a, 9, 10, Ladder]

PCR3, 9, 10 with right bands.

New PCR 1, 4a, 4b, 5, 7a with phusion

+ each assay with dNTP-mix (1µl), 5xBuffer (10µl, DMSO (1.5µl), Phusion (0.5µl) -> sum: 50µl

-> Protocol Touch down 59 with phusion, 30 cycles with gradient appropriate for the annealing temperatures above.

9-10-2010

Agarose gel electrophoresis of PCR 1, 4a, 4b, 5, 7a

-> Protocol: 11 Agarose gel electrophoresis

[From left to right: Ladder, 1, 4a, 4b, 5, 7a]

Only 4a has been amplified successfully.

Agarose gel extraction of PCR 3, 4a, 5, 9, 10

from left to right: PCR 3, band ~700bp, PCR 4a, band ~300bp, PCR 5, bands ~550bp (5*), ~650bp (5), PCR 9, band ~800bp, PCR 10, band ~1900bp

results:

-> Protocol 12 Gel extraction or PCR Clean up (Promega kit)

9-11-2010

weekend

9-12-2010

weekend

9-13-2010

charges for sequencing (retry of 6.9.)

every charge 7µl

New PCR7a with Taq

template: p190-6

Primer 13,14

Mixture

PCR program: touchdown PCR with Taq

1: 94°C 2'

2: 94°C 30"

3: 64°C/62°C/60°C/58°C/56°C/54° 30"

4: 72°C 2'

5: (for each temperature)repeat 2-4 2x

6: 94°C 30"

7: 58°C 30"

8: 72°C 2'

9: repeat 6-8 29x

10: 72°C 10'

11: 15°C break

-> Protocol: 21 PCR with Taq Mastermix

overnight culture inoculated of

CMV (amp)

ccdB (amp)

ccdB (cam)

pC31o (amp)

Bak (amp)

New PCR7a with Phusion Hot Start

with 2ng and 4 ng template

template	2ng	4ng
H2O	54µl	53µl
Buffer 5x	20µl	20µl
Primer (13,14)	2*10µl	2*10µl
DMSO	3µl	3µl
Hot Start	1µl	1µl

total: 100µl each, divided into 5 charges

program:

98°C 30sec

----

98°C 10sec

gradient: 50°C, 53°C, 56,6°C, 60,2°C, 64,5°C 30sec 30 cycles

72 15sec

----

72°C 5min

12°C forever

-> Protocol:20 PCR with Phusion Hot Start

9-14-2010

Agarose gelelectrophoresis of PCR 7a

-> Protocol: 11 Agarose gel electrophoresis

bands 1 to 5: 2ng of template DNA

bands 6 to 10: 4ng of template DNA

-> no bands

new PCR 7a and PCR 8 (without mutation) with mastermix, without DMSO

7a diluated and undiluated: divided into 3 charges

7a diluated: 1,2,3; 7a undiluated: 4,5,6

program:

94°C 2'

94°C 30"

48°C/52°C/56,1°C 30"

72°C 1,5'

(for each temperature)repeat 30x

72°C 5'

Plasmid extraction of ccdB (amp) and ccdB (cam)

-> Protocol: 4 Plasmid extraction from cells

results:

ccdB(amp): 105ng/µl A260/280: 2,00

cddB(cam): 102ng/µl A260/280: 1,952

9-15-2010

Agarose gelelectrophoresis of PCR 7a diluted &7a undiluted & "8"(without mutation)

-> Protocol: 11 Agarose gel electrophoresis

from left to right: 7a: 1:100 diluted: 48°C, 52°C, 56.1°C, undiluted: 48°C, 52°C, 56.1°C, 8 without Mutation

weak right band (with fuzz)for 7a undiluated with best result for 56°C

-> new PCR 7a and "8"

with mastermix Taq

-> Protocol: 21 PCR with Taq Mastermix

template: 190-6, 1:10 and 1:5

temp: 56°C and 58°C

2 charges each: one for 56°C and one for 58°C annealing temperature

program:

94°C 2'

94°C 30"

56°C/58°C 30"

72°C 1,5'

(for each temperature)repeat 30x

72°C 5'

Restriction Digestion of PCR 1 (17.8.), PCR 3 (10.9.), PCR 5₁ (10.9.), PCR 5₂ (10.9.), PCR 6 (17.8.), PCR 9 (10.9.), PCR 10 (30.8.) for ligation with vector

EcoR1 + Pst1 with Buffer H; 50ng DNA

-> Protocol: 5 Restriction digestion

Agarose gelelectrophoresis of PCR 7a 1-4 & "8" 1-4(without mutation)

-> Protocol: 11 Agarose gel electrophoresis

-> bad results

purification of restriction digestion

-> Protocol: 12 Gel extraction or PCR Clean up (Promega kit)

Ligation with pSB1C3 (2072bp)

plus 1 µl T4 ligase in each charge

-> Protocol: 22 Ligation

new PCR 7a & "8"

with mastermix Taq 56°C

sum: 20µl each

pcr-program:

94°C 2'

94°C 30"

56°C 30"

72°C 1,5'

(for each temperature)repeat 30x

72°C 5'

12°C forever

-> Protocol: 21 PCR with Taq Mastermix

9-16-2010

Agarose gelelectrophoresis of PCR 7a-1, PCR 7a-2, PCR 8-1, 8-2 and of all three PCR 6 we ever made (in order to control whether we have extracted the right fragment (237) or just the primerdimers)

-> Protocol: 11 Agarose gel electrophoresis

From left to right: 6₂, 6₁, 6, Ladder, 7a-1, 7a-2, 8-2, 8-1

results:

6-1 and 6-2: ?

6: ok

7a-1,7a-2,"8"-1,"8"-2: primerdimer-problem -> we ordered new primers!

transformation of the ligations 1,3,5₁, 5₂, 6, 9, 10

-> Protocol:3 Transformation

->plated on plates with chloramphenicol

again PCR 1,3,5,6,9,10 in order to increase the profit (Ausbeute) for ligation

pcr-program:

94°C 2'

94°C 30"

Tm (see above) 30"

72°C 1,5'

(for each temperature)repeat 30x

72°C 5'

-> Protocol: 21 PCR with Taq Mastermix

9-17-2010

Analysis of the transformation from yesterday

Colonies on plates when 100µl or pellet plated:

again: transformation of ligation 1

->Protocol: 3 Transformation

Agarose gelelectrophoresis of yesterday's PCR 1,3,5,6,9,10(without mutation)

-> Protocol: 11 Agarose gel electrophoresis

From left to right: PCR1, PCR3, PCR5, Ladder, PCR6, PCR9, PCR10

-> right band for PCR 3,5,6,9,10; no band for PCR 1

From left to right: Ladder, PCR3, PCR5, PCR9, PCR10

Bands took: 3₂: Band short over 1000bp, 3₁: Band short under 1000bp (probably 3₂ right), 5 the highest band, 9 upper band, 10 upper band

new PCR 1,5,6

-> Protocol: pcr-program: standard Pfu 10 PCR with Pfu

Restriction digestion of PCR products, ccdB Plasmids and Biobricks for the 3A Method

-> Protocol:5 Restriction digestion

Colony PCR of Ligations 3, 5₁, 5₂, 6, 9, 10 and PCR of CMV1 (to test if right)

10 1-4, 9 1-4, 6 1-4, 5₁ 1-4, 5₂ 1-4, 3 1-4 Colonies pickt and put in following Mix:

CMV1:

-> Protocol: 21 PCR with Taq Mastermix

Agarose Gel electrophoresis of 3, 5₁, 5₂, 6, 9, 10 and CMV1 (to test if right)

-> Protocol: 11 Agarose gel electrophoresis

Above: From left to right: 3.1, 3.2, 3.3, 3.4, 5₁.1, 5₁.2, 5₁.3, 5₁.4, Ladder, 5₂.1, 5₂.2, 5₂.3, 5₂.4

Below: From left to right: 6.1, 6.2, 6.3, 6.4, 9.1, 9.2, 9.3, 9.4, Ladder, 10.1, 10.2, 10.3, 10.4, CMV1

-> right bands for 6.3 and 6.4, CMV1 ~1200bp (we think that this is right, as Biobrick sequenzing information indicates that it isn't 654bp but about 1200bp)

PCR clean up of PCR Gel extraction 3₁, 3₂, 5, 9, 10, PCR Product 6 and digestion PCR1, Primer 18+19, R5₁, R5₂, PCR6, PCR9, PCR10, PCR3

-> Protocol: 12 Gel extraction or PCR Clean up (Promega kit)

9-18-2010

weekend

9-19-2010

weekend

9-20-2010

Dephosphorylation of linearized ccdB amp and ccdB cam

Add 1µl TSAP to digested vectors.

Incutation: 37°C 15min; Inhibition: 74°C 15min

Ligation and 3A-Assemblies

Jump-or-Die Ligations: 1a, 1b (5₁),1b (5₂), 2, 3

Cut'N'Survive Ligations: 1a, 2b,

Biobricks for both Systems: CMV, PCR1 (tet-on-promoter)

Mixtures:

- 3A Assemblies (everything except CMV, PCR1):

Inserts: each 8µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 1µl

- Biobricks:

Insert: 10µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 7µl

Incubation: 2:30h 16°C; Inhibition: 10 min 65°C

->Protocols: 22 Ligation, 13 3A Method for Biobrick assembly

Colony PCR of PCR product ligations with pSB1C3

Mixture:

PCR Mastermix:130µl ; PrimerF:19,5µl ; PrimerR:19,5µl ; H₂O:91µl

-> Protocol: 21 PCR with Taq Mastermix

Agarose Gel Electrophoresis of Colony PCRs

-> Protocol: 11 Agarose gel electrophoresis

above from left to right: pathway/ladder/3.5/3.6/3.7/3.8/5₁.5/5₁.6/5₁.7/5₁.8/5₂.5/5₂.6/5₂.7/5₂.8

below from left to right: ladder/6.5/6.6/6.7/6.8/9.5/9.6/9.7/9.8/10.5/10.6/10.7/10.8

9-21-2010

Agarose Gel Electrophoresis of PCRs 1, 5, 6, and Colony PCR 3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6

-> Bad results, probably too much cells in PCR mix

-> Protocol: 11 Agarose gel electrophoresis

Colony PCR of 3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6 again and of the 3A Ligations of yesterday as well as the Ligation of PCR1 and CMV with pSB1C3

PCR Master Mix: 60µl

Primer F: 9µl

Primer R: 9µl

H₂O: 42µl

->22charges à 5µl

-> Protocol:21 PCR with Taq Mastermix

PCR 7a with new Primers

sum: 50µl

same with Primer 13l (long)

->Protocol: 21 PCR with Taq Mastermix

Agarose Gel Electrophoresis of Colony PCRs and PCR 7a

fermentas dna ladder mix (source:http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm0333-generuler-mix

key: JD=Jump-or-Die; CS=Cut'N'Survive; first number=Ligation number (see schedule); second number=colonie number (marked on the plate)

from left to right:7ak(short primer), 7al(long primer), JD.1a.1, JD.1a.2,JD.1b5₁.1, JD.1b5₂.2, JD.1b5₂.1, JD.1b5₂.2, JD.2.1, JD.2.2, JD.3₁.1, JD.3₁.2, CS.1a.1, CS.1a.2

verified products: 7a (~800bp); JD.2.1; JD.2.2(each ~1000bp);JD.3₁.1(~2000bp); CS.1a.2(~1500bp)

no bands shown: CS.2b.1, CS.2b.2, PCR1.1, PCR1.CMV,3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6

-> Protocol: 11 Agarose gel electrophoresis

9-22-2010

Inoculate Colonies

inoculated in 4ml LB-medium with appropriate antibiotic

Plated residual transformated E. colis (9-20-10) (where we had few colonies on plates)

Colony PCRs

for 22*5µl charges:

-> Protocol: 21 PCR with Taq Mastermix

Annealing temperature: 53°C

PCR Purification of 7a

charges 7ak and 7al had not been labeled, so they were renamed.

results:

7a₁: 135ng/µl; A260/A280=1.7

7a₂: 133ng/µl; A260/A280=1.7

PCR8 with Phusion Hot Start II

-> Protocol: 20 PCR with Phusion Hot Start

Program: annealing temperature: 63°C, elongation time: 40sec

Agarose gel electrophoresis of Colony PCRs and PCR8

new ladder: fermentas FastRuler (TM) Middle Range DNA Ladder http://www.fermentas.com/en/products/all/dna-electrophoresis/fastruler-dna-ladders/sm111-fastruler-middle

some results:

PCR 8 (lanes 23, 24): no band

Colony PCRs CS.2b.10, PCR1cam.3, PCR1cam.4, CMVcam.4, CMVcam.4, JD.1b5₁.10 (lanes 7,8,9,10,11,14): show right bands

Plasmid extraction of "over-day" cultures ligation-7(=BB4),-8(=BB4),-9(=BB5),-12(=BB9) and pSB1C3

->Protocol: 4 Plasmid extraction from cells

concentrations (ng/µl - A_260/280):

BB4(7):57,5 - 1,643

BB4(8):15 - 1,5

BB5(9):173 - 1,816

BB7(12):55,0 - 1,571

pSB1C3: 12,5 - 1,250

new PCR 7b in order to get better results

overnight-cultures

inoculated of: CS2bK10=BB11; PCP1K3+4=BB1; CMV-C-K3+4; JD1b5₁K10=BB2; pSB1C3

9-23-2010

Restriction digestion of BB5 for the 3A Method

sum: 20µl

-> Protocol:5 Restriction digestion

Plasmid extraction of overnight cultures BB2, BB11, CMV in cam and pSB1C3

->Protocol: 4 Plasmid extraction from cells

concentrations (ng/µl - A_260/280):

BB2:232 - 1,603

BB11:170 - 1,889

CMV in cam:272 - 1,703

pSB1C3: 290 - 1,758

The pSB1C3 colony was pink (produced a red pigment or something like that, so we make a new transformation in order to extract only pSB1C3 and not some other vectors.

Agarose gelelectrophoresis of PCR 7b and pathway restriction digestions

-> Protocol: 11 Agarose gel electrophoresis

Gelphoto

From left to right: PCR7b, pathway (3x),ladder (5000,2000,850,400,100bp)

result: PCR 7b shows right band with ~400bp

PCR clean up of PCR 7b

-> Protocol: 12 Gel extraction or PCR Clean up (Promega kit)

result: 90ng/µl; A260/A280= 1,895

Restriction digestion of BB2 and BB11

sum: 20µl

sum: 20µl

-> Protocol 5 Restriction digestion

Ligation of BB1 and BB6

-> Protocol: 22 Ligation

PCR 8 with 7b(new) plus 7a-2 with Phusion Hot Start II; Primer 16 plus 13k and 13l

->same with Primer 13l long<-

Overnight culture of PCR1 in cam- Vector

new inoculation of PCR1 in cam, Colony 3 in 5ml LB+cam

From left to right: PCR8k,PCR8l,ladder (5000,2000,850,400,100bp)

Transformation of pSB1C3, BB1, BB6

New transformation of pSB1C3 from Spring distribution because the old colony was pink.

Transformation of BB1 and BB6.

BB1 plated on amp-Agar, BB6 and pSB1C3 plated on cam-Agar

-> Protocol: 19 Transformation (Sina Science Services)

Agarose gelelectrophoresis of PCR8 (long and short primer 13)

PCR8k=short primer 13, PCR8l=long primer 13

-> Protocol: 11 Agarose gel electrophoresis

result: PCR 8k and 8l shows right band with ~1200bp

PCR purification of PCR 8

-> Protocol: 12 Gel extraction or PCR Clean up (Promega kit)

result: ~70ng/µl; A260/A280=1.75

9-24-2010

New "over-day" culture of pSB1C3

Inoculate one culture from the ligation yesterday in 4ml LB+cam

Ligation of BB7₂

Mixture:

Inserts: each 5µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 7µl

Incubation: 5h 18°C; Inhibition: 20 min 65°C

->Protocols: 22 Ligation, 13 3A Method for Biobrick assembly

Colony PCRs of BB1 and BB6

2 Colonys were picked from each plate -> 4 charges

Mixture for 4 charges with 5µl:

+ Colony

-> Protocol: 21 PCR with Taq Mastermix

Annealing temperature: 53°C, standard protocol

Plasmid extraction of new PCR1 in cam vector

-> Protocol: 4 Plasmid extraction from cells

from left to right: 1₁, 1₂, 6₁ and 6₂

results:

Agarose Gelelektrophoresis of 1₁, 1₂, 6₁ and 6₂

Plasmid extraction of pSB1C3 in cam vector

-> Protocol: 4 Plasmid extraction from cells

results:

9-25-2010

weekend

9-26-2010

weekend

9-27-2010

Colony PCR of BB7₂, BB6 and BB1 plus a negativ charge as reference

-> Protocol 24 Colony PCR

Ligation of BB10

-> Protocol 22 Ligation

Ligation of BB001(PCR1 in ©), BB017(PCR5 in ©), BB018(PCR6 in ©) and BB021(PCR9 in ©)

-> Protocol 22 Ligation

Restriction digestion of PCR3₂, 8l, 10 and Primer 18+19 with E+P

-> Protocol 5 Restriction digestion

9-28-2010

Plating BB017, BB021, BB018, BB001, BB7₂, BB10

Colony PCR BB1, BB6, BB7₂ with DreamTaq (each 2 colonies)

-> Protocol: 16 PCR with DreamTaq

PCR mixture for PCR7a

template	pick up colony from plate
primers*2	0.25µl*2
dNTPs	0.5µl
DreamTaq	0.05µl
10xbuffer	0.5µl
H2O	3.45µl
sum	5µl

Primers for colony PCR: BBseqF, BBseqR

Annealing temperature: 53°C

Ligation of BB2, BB019, BB020, BB016, BB022

9-29-2010

plates BB7₂, BB017, BB10, BB021, BB001, BB018: nothing grown -> thrown into trash

colony-PCR of BB2/8, BB019, BB016, BB020, BB022 with Taq

->protocol: PCR with Taq

template: pick colony

sum: 10µl; we made 100µl ->10 charges (2 colonies of each plate)

tube numbers:

PCR-program: taq53

agarose-gelelektrophoresis -> no bands visible

new plates made of BB1, BB4, BB6, BB7

Ligations BB7₂, BB017 (mit PCR5₁), BB10, BB021, BB001, BB018

restriction degestions

of PCR6, PCR5₂, PCR3₂, PCR1, ccdB(amp), ccdB(cam)

->protocol: 5 Restriction digestion

colony-PCR of BB4/c3, BB6/c7, BB7₂/c5, BB1/c5, (BB11 as control) with Taq

->protocol: PCR with Taq

Mastermix:

->10µl for each charge

template: pick colony; BB11: 2ng

tube numbers:

PCR program:T_A = 53°C; elongation time=1min

9-30-2010

Colony PCR of BB2/BB8, BB6 K7&K8 and test PCR of BB11 with new dilutions and primers

-> Protocol 24 Colony PCR

PCR purification of the digestions of 6I, 6II, 5₂, 3₂, 1

-> Protocol 12 Gel extraction or PCR Clean up (Promega kit)

Transformation of BB018, BB7₂, BB001, BB017, BB10 and BB021

-> Protocol 19 Transformation SSS

Plasmidextraction of the following overnight cultures: BB020(K1), BB018(K5), BB6(K5), BB022(K1), BB016(K1), BB019(K1), BB2(K1) and BB018(K3)

-> Protocol 4 Plasmid extraction from cells

10-01-2010

new test PCR of BB5 and BB11 with 20µl and 50µl volume

Master Mix

template: BB5/50µl: 5ng, 20µl: 1.5ng; BB11/20µl: 3ng

->protocol: PCR with Taq

from left to right: ladder, BB5/50µl, BB5/20µl, BB11/20µl. BB5/50µl and BB11/20µl show the right band.

10-02-2010

weekend

10-03-2010

weekend

10-04-2010

sequencing

cycle, clean and run

PCR

10-05-2010

Colony PCRs of BB6, BB4, BB021, BB022, CMV©, BB001, BB016, BB1, BB2₅₂, BB7₂, BB017, BB018, BB019, BB020

agarose gel electrophoresis

schedule of the agarose gel electrophoresis:

Gelbilder und Auswertung einfügen

overnight cultures of BB6K3, BB4K10, CMV©K4, BB001K5, BB001K9, BB016K11, BB1K8, BB2₅₂K3, BB7₂, BB017K14, BB018K10, BB019K6

10-06-2010

Plasmidextraction of the following overnight cultures: BB6(K13), BB4(K13), CMVc (K4), BB001(K5), BB001(K9), BB016(K11), BB1(K8), BB2(K3), BB7(K10), BB017(K14), BB018(K10) and BB019(K6)

-> Protocol 4 Plasmid extraction from cells

10-07-2010

colony PCR of BB021, BB022 with Taq (53°C)

10-08-2010

plasmide extractions from 10-06-2010 were put in a lyophilizer and redissolved in less water (10-20 µl)

10-09-2010

Plasmid extraction BB018K5, BB018K6, BB021K17 and BB021K24

concentration of these samples to be measured on Monday

-> Protocol 4 Plasmid extraction from cells

10-10-2010

weekend

10-11-2010

Plasmid extraction BB018K5, BB018K6, BB021K24 and BB021K17 from 10-09-2010

Colony PCR BB01, BB018, BB6, BB252

gel picture colony PCR

foto einfügen!

Sequencing 8,9 and 10 (cycle, clean and run)

ligation BB020 BB022, Bb6 and BB5₂

10-12-2010

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10-13-2010

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10-14-2010

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10-15-2010

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10-16-2010

weekend

10-17-2010

weekend

10-18-2010

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10-19-2010

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10-20-2010

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10-21-2010

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10-22-2010

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10-23-2010

weekend

10-24-2010

weekend

10-25-2010

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10-26-2010

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10-27-2010

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10-28-2010

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10-29-2010

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10-30-2010

weekend

10-31-2010

weekend