Team:LMU-Munich/Notebook/Apoptosis

From 2010.igem.org

(Difference between revisions)
(9-15-2010)
(9-15-2010)
Line 2,937: Line 2,937:
|concentration (ng/µl)
|concentration (ng/µl)
|A<sub>260</sub>/A<sub>280</sub>
|A<sub>260</sub>/A<sub>280</sub>
-
|(supposed)lenght
+
|(supposed) lenght
|-
|-
|1
|1
Line 2,976: Line 2,976:
<font color="#009933">Ligation with pSB1C3 (2072bp)</font>
<font color="#009933">Ligation with pSB1C3 (2072bp)</font>
 +
 +
{|
 +
|-
 +
|for
 +
|#
 +
|backbone
 +
|insert (µl)
 +
|charge (µl)
 +
|Buffer 10x (µl)
 +
|Hsub<2</sub> (µl)
 +
|-
 +
|100ng
 +
|1
 +
|4µl
 +
|7,12
 +
|20
 +
|2
 +
|6,38
 +
|-
 +
|50ng
 +
|3
 +
|2
 +
|31,48
 +
|40
 +
|4
 +
|2,02
 +
|-
 +
|100ng
 +
|5<sub>1</sub>
 +
|4
 +
|8,85
 +
|20
 +
|2
 +
|4,65
 +
|-
 +
|100ng
 +
|5<sub>2</sub>
 +
|4
 +
|26,56
 +
|40
 +
|4
 +
|4,94
 +
|-
 +
|100ng
 +
|6
 +
|4
 +
|6,86
 +
|20
 +
|2
 +
|13,36
 +
|-
 +
|100ng
 +
|9
 +
|4
 +
|11,7
 +
|20
 +
|2
 +
|1,8
 +
|-
 +
|100ng
 +
|10
 +
|4
 +
|31,24
 +
|40
 +
|4
 +
|4,26
 +
|}
 +
plus 1 µl T4 ligase in each charge
 +
 +
<font color="#009933">new PCR 7a & "8")</font>
 +
 +
with mastermix Taq 56°C
 +
 +
{|
 +
|-
 +
|
 +
|7a-1
 +
|7a-2
 +
|"8"-1
 +
|"8"-2
 +
|-
 +
|mastermix (µl)
 +
|10
 +
|10
 +
|10
 +
|10
 +
|-
 +
|template
 +
|190-6 1µl
 +
|190-6 2µl
 +
|190-6(1:10) 1µl
 +
|190-6(1:10) 2µl
 +
|-
 +
|primer
 +
|13&14 2*1µl
 +
|13&14 2*1µl
 +
|13&16 2*1µl
 +
|13&16 2*1µl
 +
|-
 +
|H<sub>2</sub>O
 +
|7µl
 +
|6µl
 +
|7µl
 +
|6µl
 +
|}
 +
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
 +
|}
== 9-16-2010 ==
== 9-16-2010 ==

Revision as of 09:41, 17 September 2010


Apoptosis Notebook

Contents

Week Days
Monday Tuesday Wednesday Thursday Friday Saturday Sunday
31 8-02-2010 8-03-2010 8-04-2010 8-05-2010 8-06-2010 8-07-2010 8-08-2010
32 8-09-2010 8-10-2010 8-11-2010 8-12-2010 8-13-2010 8-14-2010 8-15-2010
33 8-16-2010 8-17-2010 8-18-2010 8-19-2010 8-20-2010 8-21-2010 8-22-2010
34 8-23-2010 8-24-2010 8-25-2010 8-26-2010 8-27-2010 8-28-2010 8-29-2010
35 8-30-2010 8-31-2010 9-01-2010 9-02-2010 9-03-2010 9-04-2010 9-05-2010
36 9-06-2010 9-07-2010 9-08-2010 9-09-2010 9-10-2010 9-11-2010 9-12-2010
37 9-13-2010 9-14-2010 9-15-2010 9-16-2010 9-17-2010 9-18-2010 9-19-2010
38 9-20-2010 9-21-2010 9-22-2010 9-23-2010 9-24-2010 9-25-2010 9-26-2010
39 9-27-2010 9-28-2010 9-29-2010 9-30-2010 10-01-2010 10-02-2010 10-03-2010

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

header 1 header 2 header 3
row 1, cell 1 row 1, cell 2 row 1, cell 3
row 2, cell 1 row 2, cell 2 row 2, cell 3

8-05-2010

this is also a table:

H2Oddes 10,3 µl
RE10 + Buffer H 2,0 µl
acetylated BSA 0,2 µl
DNA 6,0 µl


table with 3 cells

apple banana peaches
green yellow red

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

H2Oddest, sterile 10,3 µl
RE10 + Buffer H 2,0 µl
acetylated BSA (18ng/µl) 0,2 µl
DNA (0,143µg/µl) 6,0 µl

-> 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

H2Oddest, sterile 0 µl
Buffer B 2,0 µl
BSA (1:10) 2 µl
DNA (0,06µg/µl) 15,0 µl
EcoR1 0,5 µl
Spe1 0,5µl

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:

pTRERev (0,15µg/µl) pcDNA3 (0,6 µg/µl)
Primer 2*2,5µl (P1+P2) " (P11+P12)
300ng template 0,5µl 2µl
10x Buffer Pfu 5µl "
dNTP Mix 1µl "
Pfu Polymerase (3u/µl) 0,5µl "
H2O 40,5µl 39µl
summ 52,5µl 52,5µl


Programme:

Denaturation 95°C 2min
30 times: Denaturation 95°C 1min
Annealing 45°C 30sec
Extension 73°C 2min
Final Extension 73°C 5min
Soak (end) 12°C infinite

Glycerolstock of PhiC31o

- Glycerolstock of the colony of PhiC31o for the plasmidextraction

bacterial culture 800µl
Glycerol (50%) 500µl

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
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

Gelfoto from 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:

2ng 5ng 10ng
Primer 2*2,5µl (P5+P6) 2*2,5µl (P5+P6) 2*2,5µl (P5+P6)
10x Buffer Pfu 5µl 5µl 5µl
dNTP Mix 1µl 1µl 1µl
template pDS7 (dil.) 1µl 2,5µl 5µl
Pfu Polymerase (3u/µl) 0,5µl 0,5µl 0,5µl
DMSO 1,25µl 1,25µl 1,25µl
H2O 33,25µl 30,25µl 25,25µl
sum

-> 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:

ccdB tet: 50ng/µl; A260/A280= 1,818


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

PCR nr. template concentration dilution primer
7a 190-6 ~200ng/µl 1:100 13,14
7b 190-6 ~200ng/µl 1:100 15,16
9 eGFP 55ng/µl 1:25 20,21
10 PhiC31o 20ng/µl 1:10 22,23


Mixture

template (~2ng) 1µl
Pfu 0,5µl
Primer *2 2,5µl *2
10x buffer 5µl
dNTP Mix 1µl
H2O 37,5µl
sum 50µl

Standard PCR; annealing temperature: 60°C

8-26-2010

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

->Protocol: 11 Agarose gel electrophoresis

- 150V, 25min

PCR nr. expected bands result
7a 850bp no band
7b 402bp false band (200bp)
9 808bp no band
10 1888bp no band

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

template (~2ng) 1µl
Pfu 0,5µl
Primer *2 2,5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 36,25µl
sum 50µl

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

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

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

PCR nr. expected bands result
7a 850bp no band
7b 402bp right band (~400bp)+ false band (~150bp)
9 808bp false band (~200bp)
10 1888bp right band (~1900bp)+false band (~500bp)

Plasmid extraktion of ccdB amp, tet, cam

->Protocol: 4 Plasmid extraction from cells

results:

Plasmid concentration A260/A280
ccdB amp 57,5 ng/µl 1,917
ccdB cam 70,0 ng/µl 1,867
ccdB tet 50,0 ng/µl 1,818


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

template volume mass
ccdB amp 16µl 930ng
ccdB cam 14,3µl 1µg
ccdB tet 16µl 800ng
ccdB kan 16µl 400ng

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

150v, 25min, 75mA

-> Protocol: 11 Agarose gel electrophoresis

gel electrophoresis of new PCR 7a, 9, ccdb

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

template (~4ng) 2µl
Pfu 0,5µl
Primer *2 2,5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 35,25µl
sum 50µl


-> 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:

PCR nr. concentration A260/A280
7b 10 ng/µl 2,0
10 17,5 ng/µl 1,4


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

gel 9-1-10

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

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:

PCR nr. concentration A260/A280
4aP2 12,5 ng/µl 1,67
4bP2 72,5 ng/µl 1,53


New PCR 7a with Pfu and Phusion

template: 190-6, Primer 13,14

Mixture with Pfu

template (~2ng) 1µl
Pfu 0,5µl
Primer *2 2,5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 36,25µl
sum 50µl


-> 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

gel 9-2-10-2

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

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

-remain: false bands/no band


gel 9-2-10-3

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

PCR4a (~15ng) 1.2µl
PCR4b (~15ng) 2µl (1:10)
Pfu 0.5µl
Primer *2 2.5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 34.05µl
sum 50µl

-> 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

template (~2ng) 1µl
Pfu 0.5µl
Primer *2 2.5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 36.25µl
sum 50µl

-> 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

name 4a-7 4a-8 4b-9 4b-10 3-3 3-6 6-11 6-12
DNA 4a; 2.4µl 4a; 2.4µl 4b; 0.5µl 4b; 0.5µl 3; 2µl 3; 2µl 6; 0.5µl 6; 0.5µl
primer [1pmol/µl] 7; 3.2µl 8; 3.2µl 9; 3.2µl 10; 3.2µl 3; 3.2µl 6; 3.2µl 11; 3.2µl 12; 3.2µl
Tris (10mM); pH 8.2 1.4µl 1.4µl 3.3µl 3.3µl 1.8µl 1.8µl 3.3µl 3.3µl

every charge 7µl


New overlapping PCR 5 with Pfu

template: 4a, 4b; 15ng

Primer 7,10


Mixture

PCR4a (~7ng) 0.56µl
PCR4b (~8ng) 1.1µl (1:10)
Pfu 0.5µl
Primer *2 2.5µl *2
10x buffer 5µl
dNTP Mix 1µl
DMSO 1,25µl
H2O 36.6µl
sum 50µl

-> 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

template[2ng/µl] 1µl 2µl
pfu polymerase 0.5µl 0.5µl
primer13 2.5µl 2.5µl
primer14 2.5µl 2.5µl
buffer 5µl 5µl
dNTP Mix 1µl 1µl
DMSO 1,25µl 1.25µl
H2O 36.25µl 35.25
sum 50µl 50µl


Agarose gel electrophoresis of 7a

-> results: "primer clouds"


charges for sequencing for PCR 1,7b and 9

name 1-1 1-2 7b-15 7b-16 9-20 9-21 10-22 10-23
DNA 1[1:10]; 1.74µl 1[1:10]; 1.74µl 7b; 3µl 7b; 3µl 9; 1.78µl 9; 1.78µl 10; 3.8µl 10; 3.8µl
primer [1pmol/µl] 1; 3.2µl 2; 3.2µl 15; 3.2µl 16; 3.2µl 20; 3.2µl 21; 3.2µl 22; 3.2µl 23; 3.2µl
Tris (10mM); pH 8.2 2.06µl 2.06µl 0.8µl 0.8µl 2.02µl 2.02µl 0µl 0µl

every charge 7µl

9-07-2010

Agarose gel electrophorese of PCR gel extractions

150V, 25min

-> Protocol: 11 Agarose gel electrophoresis

-result:

gel photo

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

PCR nr. 1 2a 2b 3 4a 7b 9 10
expected band (bp) 492 332 772 1087 330 402 808 1888
shown band(s) 550,200 300 750 1100 300 450 900,1500 1900
clean charge x x ~x x x x

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

eGFP SV40PA=PCR6 ccdBamp
DNA 5µl=300ng DNA 1µl=37,5ng DNA 2µl=115ng
Buffer MC 2µl Buffer D 2µl Buffer H 2µl
BSA 1:10 2µl BSA 1:10 2µl BSA 1:10 2µl
EcoRI 0,5µl XbaI 0,5µl EcoRI 0,5µl
SpeI 0,5µl PstI 0,5µl PstI 0,5µl
H2O 13µl H2O 14µl H2O 13µl
sum 23µl sum 20µl sum 20µl

1:30h 37°C, 20min 80°C

Agarose gel electrophoresis of PCR6

-> Protocol: 11 Agarose gel electrophoresis

25min, 150V


Ligation 1

eGFP 14,4µL (144ng)
SV40PA 4,8µL (48ng)
ccdBamp 2,9µL (100ng)
T4 Buffer 10x 3µL
T4 Ligase 0,5µL
H2O 4,4µL
sum 30µL


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

Concentration of DNA in Ligation 1:

540ng A260/A280=2,4


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

Plasmid concentration [ng/µL] A260/A280
ccdBcam 22.5 1.0
ccdBamp 42.5 1.2
Bak 17.5 0.8
CMV 10.0 0.7
PhiC310 60 1.4

Eluated with H2O instead of the Eluation Buffer.


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

PCRnr. 1 3 4a 4b 5 7a 9 10
template pDS7 (1:100); 4µl 2a; 0.9µl+2b; 0.6µl Bak; 0.5µl Bak; 0.5µl 4a; 0.8µl+4b; 1µl 190-6 (1:100); 1µl eGFP (1:25); 2µl PhiC31o (1:10); 1µl
primer [10pmol/µl] 1,2; 2.5µl 3,6; 2.5µl 7,8; 2.5µl 9,10; 2.5µl 7,10; 2.5µl 13,14; 2.5µl 20,21; 2.5µl 22,23; 2.5µl
H2O 28µl 30.5µl 31.5µl 31.5µl 30.2µl 31µl 30µl 31µl
Annealing temperature 51.5°C 53.1°C 53.1°C 51.5°C 53.1°C 57.5°C 61°C 57.5°C

+ 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

gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9, 10

[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

PCRnr. 1 4a 4b 5 7a
template pDS7 (1:10); 1µl Bak 1 ; 1µl Bak 1; 1µl 4a, 4b; 2 x 1.5µl pCT 190-6 (1:40); 1µl
primer 1, 2; 2 x 2.5µl 7, 8; 2 x 2.5µl 9, 10; 2 x 2.5µl 7, 10; 2 x 2.5µl 13, 14; 2x 2.5µl
H2O 31µl 31µl 31µl 30µl 30µl
Annealing temperature 52°C 52°C 48°C 50°C 55°C

+ 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

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

[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


gel electrophoresis 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:

PCR 3 PCR 4a PCR 5 PCR 5* PCR 9 PCR 10
concentration [ng/µl] 20 10 5 30 35 25
A260/A280 1.6 1.333 2.0 2.0 1.750 2.0


-> 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.)

name 4a-7 4a-8 4b-9 4b-10 3-3 3-6 6-11 6-12
DNA 4a; 2.4µl 4a; 2.4µl 4b; 0.5µl 4b; 0.5µl 3; 2µl 3; 2µl 6; 0.5µl 6; 0.5µl
primer [1pmol/µl] 7; 3.2µl 8; 3.2µl 9; 3.2µl 10; 3.2µl 3; 3.2µl 6; 3.2µl 11; 3.2µl 12; 3.2µl
Tris (10mM); pH 8.2 1.4µl 1.4µl 3.3µl 3.3µl 1.8µl 1.8µl 3.3µl 3.3µl

every charge 7µl


New PCR7a with Taq

template: p190-6

Primer 13,14

Mixture

template (~4ng) 2µl (1:100)
MasterMix for Taq 10µl
Primer *2 1.5µl *2
DMSO 0,5µl
H2O 4.5µl
sum 20µl

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


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

9-14-2010

Agarose gelelectrophoresis of PCR 7a


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

number: 7a diluted 7a undiluted 8
mastermix 50µl 50µl 10µl
primer 2*10µl 2*10µl 2*2µl
H2O 29µl 29µl 5µl
template 1µl 190-6 (1:100) 1µl 190-6 1µl 190-6 (1:100)

7a diluated and undiluated: divided into 3 charges

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

1 2 3 4 5 6 8
48°C 52°C 56,1°C 48°C 52°C 56,1°C 52°C

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)

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)

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

-> new PCR 7a and "8"

with mastermix Taq

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

temp: 56°C and 58°C


number 7a 7a "8" "8"
Mastermix 20µl 20µl 20µl 20µl
H2O 15µl 15µl 15µl 15µl
Primer (13,14) 2*2µl (13,14) 2*2µl (13,16) 2*2µl (13,16) 2*2µl
Template 190-6 (1:5) 1µl 190-6 (1:10) 1µl 190-6 (1:5) 1µl 190-6 (1:10) 1µl

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 vektor

EcoR1 + Pst1 with Buffer H; 50ng DNA

1 3 51 52 6 9 10
H2O 13µl 12,5µl 5µl 13µl 13,5µl 13,5µl 12,5µl
Buffer H 2µl 2µl 2µl 2µl 2µl 2µl 2µl
BSA (1:10) 2µl 2µl 2µl 2µl 2µl 2µl 2µl
DNA 2µl 2,5µl 10µl 2µl 1,5µl 1,5µl 2,5µl
EcoR1 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl
Pst1 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl 0,5µl

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

-> bad results

purification and restriction digestion

concentration (ng/µl) A260/A280 (supposed) lenght
1 20 1,000 492
3 5 1,000 1087
51 22,5 1,8 688
52 7,5 1,5 688
6 10 1,333 237
9 20 1,6 808
10 17,5 1,75 1888

Ligation with pSB1C3 (2072bp)

for # backbone insert (µl) charge (µl) Buffer 10x (µl) Hsub<2</sub> (µl)
100ng 1 4µl 7,12 20 2 6,38
50ng 3 2 31,48 40 4 2,02
100ng 51 4 8,85 20 2 4,65
100ng 52 4 26,56 40 4 4,94
100ng 6 4 6,86 20 2 13,36
100ng 9 4 11,7 20 2 1,8
100ng 10 4 31,24 40 4 4,26

plus 1 µl T4 ligase in each charge

new PCR 7a & "8")

with mastermix Taq 56°C

7a-1 7a-2 "8"-1 "8"-2
mastermix (µl) 10 10 10 10
template 190-6 1µl 190-6 2µl 190-6(1:10) 1µl 190-6(1:10) 2µl
primer 13&14 2*1µl 13&14 2*1µl 13&16 2*1µl 13&16 2*1µl
H2O 7µl 6µl 7µl 6µl

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

9-16-2010

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9-18-2010

weekend

9-19-2010

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9-20-2010

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9-25-2010

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9-26-2010

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9-27-2010

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10-02-2010

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10-03-2010

weekend