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Apoptosis Notebook

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:

H₂O_ddes	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

H₂O_{ddest, 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

- 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

- 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

- restriction digest of PhiC310 with EcoR1 and Spe1

H₂O_{ddest, 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")

-Primer handling after arrival

- 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)

Primer	2*2,5µl
300ng template	pcDNA3 (0,6 µg/µl) 0,5µl	pTRERev (0,15µg/µl) 2µl
10x Buffer Pfu	5µl
dNTP Mix	1µl
Pfu Polymerase (3u/µl)	0,5µl
H₂O	pcDNA3 40,5µl	pTRERev 39µl
summ	52,5µl	52,5µl 8-17-2010 text 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

@@ Line 268: / Line 268: @@
 -Primer handling after arrival
+- 10mM dNTP mix made from 100 mM dATP, dGTP, dCTP, dTTP by taking 100µl of each and adding 600µl H <sub>2</sub> 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)
+{|
+|Primer
+|2*2,5µl
+|-
+|300ng template
+|pcDNA3 (0,6 µg/µl) 0,5µl
+|pTRERev (0,15µg/µl) 2µl
+|-
+|10x Buffer Pfu
+|5µl
+|-
+|dNTP Mix
+|1µl
+|-
+|Pfu Polymerase (3u/µl)
+|0,5µl
+|-
+|H<sub>2</sub>O
+|pcDNA3 40,5µl
+|pTRERev 39µl
+|-
+|summ
+|52,5µl
+|52,5µl
 == 8-17-2010 ==

Team:LMU-Munich/Notebook/Apoptosis