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Apoptosis Notebook

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
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 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, 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 extrction

- 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

- 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

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")

->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

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

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

-> Protocol (4 Plasmid extraction from cells)

- Restriction digest 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

-> Protocol (5 Restriction digest)

- 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 16.8)

8-19-2010

- Agarose gel electrophoresis of PCR 2a and 2b (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)

-> Protocol (11 Agarose gel electrophoresis)

- 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

weekend

10-03-2010

weekend

@@ Line 244: / Line 244: @@
 == 8-12-2010 ==
--     plasmid extraction of pDS7 (458ng/µl), eGFP (55ng/µl), 190-6 (193ng/µl)
+<font color="#009933">Plasmid Extraction of pDS7, eGFP, 190-6</font>
 ->	Protocol: ([[Team:LMU-Munich/Notebook/Protocols/4_Plasmid_extraction_from_cells|4 Plasmid extraktion from cells]])
--	Restriction digest with EcoRI and PstI in buffer H (for testing DNA is correct)
+-       pDS7 (458ng/µl), eGFP (55ng/µl), 190-6 (193ng/µl)
+<font color="#009933">Restriction digest of pDS7, eGFP, 190-6</font>
+-	with EcoRI and PstI in buffer H (for testing DNA is correct)
+->      Protocol: ([[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digest]])
-µg DNA: pDS7 (2µl), eGFP (15µl), 190-6 (10µl)
+-       10µg DNA: pDS7 (2µl), eGFP (15µl), 190-6 (10µl)
-->      Protocol: ([[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digest]])
+<font color="#009933">Plate colonies for plasmid extrction</font>
--	Colony for Plasmidextraction (CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin)) plated
+-	CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin))
 -	PhiC31o plated on Ampicillin-Agar, stored at 37°C
--	50% Glycerol made (for the glycerolstock PhiC31o)
+<font color="#009933">50% Glycerol made</font>
+-       for PhiC31o glycerol stock (produced later)
 == 8-13-2010 ==

Team:LMU-Munich/Notebook/Apoptosis

From 2010.igem.org

Revision as of 08:15, 20 August 2010

Apoptosis Notebook

Contents

8-02-2010

8-03-2010

8-04-2010

8-05-2010

8-06-2010

8-07-2010

8-08-2010

8-09-2010

8-10-2010

8-11-2010

8-12-2010

8-13-2010

8-14-2010

8-15-2010

8-16-2010

8-17-2010

8-18-2010

8-19-2010

8-20-2010

8-21-2010

8-22-2010

8-23-2010

8-24-2010

8-25-2010

8-26-2010

8-27-2010

8-28-2010

8-29-2010

8-30-2010

8-31-2010

9-01-2010

9-02-2010

9-05-2010

9-04-2010

9-05-2010

9-06-2010

9-07-2010

9-08-2010

9-09-2010

9-10-2010

9-11-2010

9-12-2010

9-13-2010

9-14-2010

9-15-2010

9-16-2010

9-17-2010

9-18-2010

9-19-2010

9-20-2010

9-21-2010

9-22-2010

9-23-2010

9-24-2010

9-25-2010

9-26-2010

9-27-2010

9-28-2010

9-29-2010

9-30-2010

10-01-2010

10-02-2010

10-03-2010