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Apoptosis Notebook

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 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 digestion 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

Inoculate CMV into LB medium with amicillin

- 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

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", 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

-> 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:

	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) CMV (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 resistence on LB agar with appropiate antibiotic.

Overnight culture of ccdB kan

- Overnight culture of ccdB with kanamycine resistence 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

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

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

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

- 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

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

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

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

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

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:

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


	30µL

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

Concentration of DNA in Ligation 1:

weekend

10-03-2010

weekend

@@ Line 2,315: / Line 2,315: @@
 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
+|}
+<font color="#009933">Transformation</font>
 == 9-09-2010 ==

Team:LMU-Munich/Notebook/Apoptosis

From 2010.igem.org

Revision as of 14:41, 8 September 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

9-03-2010

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