Team:LMU-Munich/Notebook/Apoptosis

From 2010.igem.org

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== 8-17-2010 ==
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[[Image:17 8 10_1_apo.jpg|thumb|right|Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6]]
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[[Image:17 8 10_2_apo.jpg|thumb|right|Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6 which shows that PCR1 is between 250 and 500 bp]]
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[[Image:17.8.2010 PCR6.jpg|thumb|right|Agarose gel electrophoresis of PCR6 which shows that PCR6 is about 200bp]]
[[Image:17.8.2010 PCR6.jpg|thumb|right|Agarose gel electrophoresis of PCR6 which shows that PCR6 is about 200bp]]
- colonies for plasmidextraction of CMV and pDS7 plated on Ampicillinplates
- colonies for plasmidextraction of CMV and pDS7 plated on Ampicillinplates
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- no band found for PCR6; new electrophoresis needed with more DNA loaded
- no band found for PCR6; new electrophoresis needed with more DNA loaded
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Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6
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Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6 which shows that PCR1 is between 250 and 500 bp
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- new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown)
- new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown)

Revision as of 18:45, 17 August 2010


Apoptosis Notebook

Contents

Week Days
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

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 too is 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 digest (Restriktionsverdau) 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 (458ng/µl), eGFP (55ng/µl), 190-6 (193ng/µl)

-> Protocol: (4 Plasmid extraktion from cells)

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

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

-> Protocol: (5 Restriction digest)

- Colony for Plasmidextraction (CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin)) plated

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

- 50% Glycerol made (for the glycerolstock PhiC31o)

8-13-2010

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

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

- 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

->Protocol (11 Agarose gel electrophoresis)

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

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

-> Protocol (5 Restriction digest)

- Agarosegelelectorphoresis for 30 minutes, 150V

- 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

->Protocol (11 Agarose gel electrophoresis)

- 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

- 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 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

->Protocol (4 Plasmid extraktion from cells)


- 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")

->Protocol (5 Restriction digest)


- Primer handling after arrival

->Protocol (9 Handling primers)

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

-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)


Mixture:

pcDNA3 (0,6 µg/µl) pTRERev (0,15µg/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

->Protocol (10 PCR with Pfu)


- 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

- colonies for plasmidextraction of CMV and pDS7 plated on Ampicillinplates

- 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 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)

->Protocol (11 Agarose gel electrophoresis)

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

- 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)

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

8-18-2010

Next iGEM meeting at 6pm!

8-19-2010

Workshop with Tanya on Wiki/Web page at 6pm

8-20-2010

text

8-21-2010

text

8-22-2010

text