Team:LMU-Munich/Notebook/Apoptosis

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Latest revision as of 18:04, 27 October 2010


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ApoControl Notebook

What we did

Short description of our work, our results and our supporters

The creation of certain constructs was necessary for our two systems for cell selection by means of apoptosis: “Cut’N’Survive” and “Jump-Or-Die”. We searched for sources of the DNA sequences we needed and found several supporters which are listed below.

Most genes and promoters were amplificated via PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. All PCRs worked out, even the fusion PCRs.

The length of the PCR products were tested by agarose gel electrophoresis. We tried to sequence our PCR products, but obtained poor results and resorted to sequencing the plasmids.

In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were digested with the appropriate restriction enzymes. The samples were cleaned with a PCR clean up kit or dephosphorylated to reduce false ligations.

We ligated our constructs and several interim stages with the 3A-assembly according to our schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. Afterwards, some colonies were picked and we tested the insertion of the construct by colony PCR.

If the colony PCR resulted in bands of the right size, we extracted the plasmids from overnight cultures and sequenced the samples with forward and reverse BioBrick primers.

Unfortunately, not all BioBricks were cloned succesfully. However, we were able to produce 4 BioBricks, one of which represents a full construct while the other three are intermediates. The system wasn't completed on time, so we weren´t able to test them in eukarytic cell lines.

The protocols we used are listed here: Protocols

These Biobricks we submitted:

BBa_K368004: attP+eGFP+SV40PA
BBa_K368011: eGFP+SV40PA
BBa_K368016: TEVrecognition site+N-degron+SF3b155
BBa_K368019: TEV-Protease+p14*+TEVrecognition site

Sources, helpers and supporters:

Prof. Dr. Angelika Böttger :
- prevTRE (tet-on CMV promoter; inducible by doxycycline in special cell lines)
- supported the construction ideas and would have given us the cells and mediums we would have needed
- SV40PA (Polyadenylation site): gave us a vector containing it
- Human Bak: her assistant Erika Clement gave us appropriate cDNA

Dr. Arnim Weber: submitted us a vector with human Bak
Dr. Philipe Soriano: (Raymond CS et al: High-Efficiency FLP and PhiC31 Site-Specific Recombination in Mammalian Cells (2007))
- Sequences of attB and attP site
- PhiC31o was bought via addgene
Knop, M (Heidelberg): (Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009))
- TEVrecognition site+N-degron+SF3b155
- TEV-Protease+p14*+TEVrecognition site
Prof. Dr. Thorsten Mascher:
- Helped with primer design, agarose gel electrophoresis apparatuses and trouble shooting
Prof. Dr. Kirsten Jung:
- Helped with ideas and fundraising
Dr. Susanne Gebhard:
- Helped with trouble-shooting and materials
Prof. Dr. Andreas Brachmann:
- Sequenced our samples
Partsregistry:
- eGFP (BBa_I714891)
- CMV-Promoter (BBa_J52034: this part was wrong: its lacI !!!)
- ccdB amp, cam, tet, kan in E.coli DH3

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
40	10-04-2010	10-05-2010	10-06-2010	10-07-2010	10-08-2010	10-09-2010	10-10-2010
41	10-11-2010	10-12-2010	10-13-2010	10-14-2010	10-15-2010	10-16-2010	10-17-2010
42	10-18-2010	10-19-2010	10-20-2010	10-21-2010	10-22-2010	10-23-2010	10-24-2010
43	10-25-2010	10-26-2010	10-27-2010	10-28-2010	10-29-2010	10-30-2010	10-31-2010

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 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 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

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; rightmost 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

- Quiagen kit (QuiaexII)

-> 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

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; 4b: 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
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

[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

-> Protocol: 11 Agarose gel electrophoresis

[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

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

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

-> Protocol: 21 PCR with Taq Mastermix

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

-> Protocol:20 PCR with Phusion Hot Start

9-14-2010

Agarose gelelectrophoresis of PCR 7a

-> Protocol: 11 Agarose gel electrophoresis

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)

-> Protocol: 4 Plasmid extraction from cells

results:

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)

-> Protocol: 11 Agarose gel electrophoresis

from left to right: 7a: 1:100 diluted: 48°C, 52°C, 56.1°C, undiluted: 48°C, 52°C, 56.1°C, 8 without Mutation

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

-> new PCR 7a and "8"

with mastermix Taq

-> Protocol: 21 PCR with Taq Mastermix

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 vector

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

-> Protocol: 5 Restriction digestion

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

-> Protocol: 11 Agarose gel electrophoresis

-> bad results

purification of 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

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

Ligation with pSB1C3 (2072bp)

for	#	backbone	insert (µl)	charge (µl)	Buffer 10x (µl)	H₂O (µl)
100ng	1	4	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

-> Protocol: 22 Ligation

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

-> Protocol: 21 PCR with Taq Mastermix

9-16-2010

Agarose gelelectrophoresis of PCR 7a-1, PCR 7a-2, PCR 8-1, 8-2 and of all three PCR 6 we ever made (in order to control whether we have extracted the right fragment (237) or just the primerdimers)

-> Protocol: 11 Agarose gel electrophoresis

From left to right: 6₂, 6₁, 6, Ladder, 7a-1, 7a-2, 8-2, 8-1

results:

6-1 and 6-2: ?

6: ok

7a-1,7a-2,"8"-1,"8"-2: primerdimer-problem -> we ordered new primers!

transformation of the ligations 1,3,5₁, 5₂, 6, 9, 10

-> Protocol:3 Transformation

->plated on plates with chloramphenicol

again PCR 1,3,5,6,9,10 in order to increase the profit (Ausbeute) for ligation

	1	3	5	6	9	10
template	prev TRE(1:30) 1µl	2a(1:5) 1µl + 2b 0,5µl	4a 0,5µl + 4b 0,5µl	SV40PA (pcDNA3)(1:50) 1µl	eGFP(1:5) 1µl	PhiC310(1:6) 1µl
primer	1&2 2*2,5µl	3&6 2*2,5µl	7&10 2*2,5µl	11&12 2*2,5µl	20&21 2*2,5µl	22&23 2*2,5µl
T_m	54°C	50°C	49°C	56°C	58°C	58°C
mastermix (µl)	25	25	25	25	25	25
DMSO	1,25µl	1,25µl	1,25µl	1,25µl	1,25µl	1,25µl
H₂O (µl)	17,75	17,25	17,75	17,75	17,75	17,75

pcr-program:

94°C 2'

94°C 30"

T_m (see above) 30"

72°C 1,5'

(for each temperature)repeat 30x

72°C 5'

-> Protocol: 21 PCR with Taq Mastermix

9-17-2010

Analysis of the transformation from yesterday

Colonies on plates when 100µl or pellet plated:

	1	3	5	6	9	10
pellet	no	yes	yes	yes	yes	yes	yes
100µl	no	yes	yes	yes	yes	yes	yes

again: transformation of ligation 1

->Protocol: 3 Transformation

Agarose gelelectrophoresis of yesterday's PCR 1,3,5,6,9,10(without mutation)

-> Protocol: 11 Agarose gel electrophoresis

From left to right: PCR1, PCR3, PCR5, Ladder, PCR6, PCR9, PCR10

-> right band for PCR 3,5,6,9,10; no band for PCR 1

From left to right: Ladder, PCR3, PCR5, PCR9, PCR10

Bands took: 3₂: Band short over 1000bp, 3₁: Band short under 1000bp (probably 3₂ right), 5 the highest band, 9 upper band, 10 upper band

new PCR 1,5,6

	1	5	6
template	pTRE Rev 0,5µl	4a 1,5µl + 4b 1,5µl	pcDNA3 0,5µl
primer	1&2 2*2,5µl	7&10 2*2,5µl	11&12 2*2,5µl
buffer 10x	5µl	5µl	5µl
dNTPs	1µl	1µl	1µl
Pfu	0,5µl	0,5µl	0,5µl
H₂O	26,75 µl	24,25 µl	26,75 µl
T<sub<m</sub>	51°C	50°C	55°C

-> Protocol: pcr-program: standard Pfu 10 PCR with Pfu

Restriction digestion of PCR products, ccdB Plasmids and Biobricks for the 3A Method

Name	H₂O	Buffer (each 2µl)	BSA (1:10)	DNA Volume	DNA Mass	Enzyms (2*0.5µl)
ccdBa	25 µl	H	2µl	20µl	~600ng	E+P
ccdBa	25µl	H	2µl	20µl	~700ng	E+P
1	43µl	MC	2µl	2µl	~500ng	E+S
3	25µl	B	2µl	20µl	~200ng	X+P
Primer 18+19	25µl	B	2µl	10µl+10µl	?	X+P
5₁	25µl	MC	2µl	20µl	~100ng	E+S
5₂	25µl	MC	2µl	20µl	~600ng	E+S
6	35µl	B	2µl	10µl	~400ng	X+P
9	25µl	MC	2µl	20µl	~700ng	E+S
10	25µl	MC	2µl	20µl	~400ng	E+S
eGFP	35µl	MC	2µl	10µl	~550ng	E+S
CMV1	35µl	H	2µl	10µl	~530ng	E+P

-> Protocol:5 Restriction digestion

Colony PCR of Ligations 3, 5₁, 5₂, 6, 9, 10 and PCR of CMV1 (to test if right)

10 1-4, 9 1-4, 6 1-4, 5₁ 1-4, 5₂ 1-4, 3 1-4 Colonies picked and put in following Mix:

PCR Mastermix	10µl
Biobrick Primer F	1.5µl
Biobrick Primer R	1.5µl
H₂O	7µl

CMV1:

PCR Mastermix	10µl
Biobrick Primer F	1.5µl
Biobrick Primer R	1.5µl
Template	2µl
H₂O	5µl

-> Protocol: 21 PCR with Taq Mastermix

Agarose Gel electrophoresis of 3, 5₁, 5₂, 6, 9, 10 and CMV1 (to test if right)

-> Protocol: 11 Agarose gel electrophoresis

Above: From left to right: 3.1, 3.2, 3.3, 3.4, 5₁.1, 5₁.2, 5₁.3, 5₁.4, Ladder, 5₂.1, 5₂.2, 5₂.3, 5₂.4

Below: From left to right: 6.1, 6.2, 6.3, 6.4, 9.1, 9.2, 9.3, 9.4, Ladder, 10.1, 10.2, 10.3, 10.4, CMV1

-> right bands for 6.3 and 6.4, CMV1 ~1200bp (we think that this is right, as Biobrick sequenzing information indicates that it isn't 654bp but about 1200bp)

PCR clean up of PCR Gel extraction 3₁, 3₂, 5, 9, 10, PCR Product 6 and digestion PCR1, Primer 18+19, R5₁, R5₂, PCR6, PCR9, PCR10, PCR3

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

9-18-2010

weekend

9-19-2010

weekend

9-20-2010

Dephosphorylation of linearized ccdB amp and ccdB cam

Add 1µl TSAP to digested vectors.

Incutation: 37°C 15min; Inhibition: 74°C 15min

Ligation and 3A-Assemblies

Jump-or-Die Ligations: 1a, 1b (5₁),1b (5₂), 2, 3

Cut'N'Survive Ligations: 1a, 2b,

Biobricks for both Systems: CMV, PCR1 (tet-on-promoter)

Mixtures:

- 3A Assemblies (everything except CMV, PCR1):

Inserts: each 8µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 1µl

- Biobricks:

Insert: 10µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 7µl

Incubation: 2:30h 16°C; Inhibition: 10 min 65°C

->Protocols: 22 Ligation, 13 3A Method for Biobrick assembly

Colony PCR of PCR product ligations with pSB1C3

Mixture:

PCR Mastermix:130µl ; PrimerF:19,5µl ; PrimerR:19,5µl ; H₂O:91µl

-> Protocol: 21 PCR with Taq Mastermix

Agarose Gel Electrophoresis of Colony PCRs

-> Protocol: 11 Agarose gel electrophoresis

above from left to right: pathway/ladder/3.5/3.6/3.7/3.8/5₁.5/5₁.6/5₁.7/5₁.8/5₂.5/5₂.6/5₂.7/5₂.8

below from left to right: ladder/6.5/6.6/6.7/6.8/9.5/9.6/9.7/9.8/10.5/10.6/10.7/10.8

9-21-2010

Agarose Gel Electrophoresis of PCRs 1, 5, 6, and Colony PCR 3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6

-> Bad results, probably too much cells in PCR mix

-> Protocol: 11 Agarose gel electrophoresis

Colony PCR of 3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6 again and of the 3A Ligations of yesterday as well as the Ligation of PCR1 and CMV with pSB1C3

PCR Master Mix: 60µl

Primer F: 9µl

Primer R: 9µl

H₂O: 42µl

->22charges à 5µl

-> Protocol:21 PCR with Taq Mastermix

PCR 7a with new Primers

template 190-6 (1:10)	1µl
Master Mix	25µl
Primer 13k (short)	3,75µl
Primer 14	3,75µl
H₂O	16,5µl

sum: 50µl

same with Primer 13l (long)

->Protocol: 21 PCR with Taq Mastermix

Agarose Gel Electrophoresis of Colony PCRs and PCR 7a

different ladder:

fermentas dna ladder mix (source:http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm0333-generuler-mix

key: JD=Jump-or-Die; CS=Cut'N'Survive; first number=Ligation number (see schedule); second number=colonie number (marked on the plate)

from left to right:7ak(short primer), 7al(long primer), JD.1a.1, JD.1a.2,JD.1b5₁.1, JD.1b5₂.2, JD.1b5₂.1, JD.1b5₂.2, JD.2.1, JD.2.2, JD.3₁.1, JD.3₁.2, CS.1a.1, CS.1a.2

verified products:7a (~800bp); JD.2.1; JD.2.2(each ~1000bp);JD.3₁.1(~2000bp); CS.1a.2(~1500bp)

no bands shown: CS.2b.1, CS.2b.2, PCR1.1, PCR1.CMV,3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6

-> Protocol: 11 Agarose gel electrophoresis

9-22-2010

Inoculate Colonies

inoculated in 4ml LB-medium with appropriate antibiotic

Plated residual transformated E. colis (9-20-10) (where we had few colonies on plates)

Colony PCRs

for 22*5µl charges:

PCR Mastermix	55µl
Primer F	8,25µl
Primer R	8,25µl
H₂O	38,5µl
sum	110µl

-> Protocol: 21 PCR with Taq Mastermix

Annealing temperature: 53°C

PCR Purification of 7a

charges 7ak and 7al had not been labeled, so they were renamed.

results:

7a₁: 135ng/µl; A260/A280=1.7

7a₂: 133ng/µl; A260/A280=1.7

PCR8 with Phusion Hot Start II

-> Protocol: 20 PCR with Phusion Hot Start

template 7a 1:100	3µl ~4ng (850bp)
template 7b 1:10	2µl ~2ng (402bp)
Buffer 5X	10µl
Phusion	0.5µl
H₂O	27µl
Primer 13	2.5µl
Primer 16	2.55µl
DMSO)	1.5µl
dNTP mix	1µl

Program: annealing temperature: 63°C, elongation time: 40sec

Agarose gel electrophoresis of Colony PCRs and PCR8

new ladder: fermentas FastRuler (TM) Middle Range DNA Ladder http://www.fermentas.com/en/products/all/dna-electrophoresis/fastruler-dna-ladders/sm111-fastruler-middle

some results:

PCR 8 (lanes 23, 24): no band

Colony PCRs CS.2b.10, PCR1cam.3, PCR1cam.4, CMVcam.4, CMVcam.4, JD.1b5₁.10 (lanes 7,8,9,10,11,14): show right bands

Plasmid extraction of "over-day" cultures ligation-7(=BB4),-8(=BB4),-9(=BB5),-12(=BB9) and pSB1C3

->Protocol: 4 Plasmid extraction from cells

concentrations (ng/µl - A_260/280):

BB4(7):57,5 - 1,643

BB4(8):15 - 1,5

BB5(9):173 - 1,816

BB7(12):55,0 - 1,571

pSB1C3: 12,5 - 1,250

new PCR 7b in order to get better results

Master Mix	25µl
H₂O	16,5µl
Primer 15	3,75µl
Primer 16	3,75µl
template 190-6 (1:10)	1µl

overnight-cultures

inoculated of: CS2bK10=BB11; PCP1K3+4=BB1; CMV-C-K3+4; JD1b5₁K10=BB2; pSB1C3

9-23-2010

Restriction digestion of BB5 for the 3A Method

Name	H₂O	Buffer D	BSA (1:10)	DNA Volume	DNA Mass	Enzyms (2*0.5µl)
BB5	11 µl	2µl	2µl	4µl	~500ng	X+P

sum: 20µl

-> Protocol:5 Restriction digestion

Plasmid extraction of overnight cultures BB2, BB11, CMV in cam and pSB1C3

->Protocol: 4 Plasmid extraction from cells

concentrations (ng/µl - A_260/280):

BB2:232 - 1,603

BB11:170 - 1,889

CMV in cam:272 - 1,703

pSB1C3: 290 - 1,758

The pSB1C3 colony was pink (produced a red pigment or something like that, so we make a new transformation in order to extract only pSB1C3 and not some other vectors.

Agarose gelelectrophoresis of PCR 7b and pathway restriction digestions

-> Protocol: 11 Agarose gel electrophoresis

Gelphoto

From left to right: PCR7b, pathway (3x),ladder (5000,2000,850,400,100bp)

result: PCR 7b shows right band with ~400bp

PCR clean up of PCR 7b

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

result: 90ng/µl; A260/A280= 1,895

Restriction digestion of BB2 and BB11

Name	H₂O	Buffer D	BSA (1:10)	DNA Volume	DNA Mass	Enzyms (2*0.5µl)
BB2	10 µl	2µl	2µl	5µl	~1.2mg	X+P

sum: 20µl

Name	H₂O	Buffer D	BSA (1:10)	DNA Volume	DNA Mass	Enzyms (2*0.5µl)
BB11	7 µl	2µl	2µl	8µl	~1.3mg	X+P

sum: 20µl

-> Protocol 5 Restriction digestion

Ligation of BB1 and BB6

-> Protocol: 22 Ligation

Name	H₂O	T4 Ligase-Buffer	PCR1	Primer 18+19	ccdBAmp	T4 Ligase
BB1	9.5 µl	2µl	5µl	2µl	1µl	0.5µl

Name	H₂O	T4 Ligase-Buffer	CMV E+S	BB5	ccdBCam	T4 Ligase
BB6	6.5 µl	2µl	8µl	2µl	1µl	0.5µl

PCR 8 with 7b(new) plus 7a-2 with Phusion Hot Start II; Primer 16 plus 13k and 13l

Buffer 5x	10µl
H₂O	27µl
dNTPs	1µl
Primer 16	2,5µl
Primer 13k short	2,5µl
DMSO	1,5µl
template 7a(1:100)(850bp)	3µl -> 4ng
template 7b(new)(1:100)(402bp)	2µl -> 2ng
Phusion Hot Start II	0,5µl

->same with Primer 13l long<-

Overnight culture of PCR1 in cam- Vector

new inoculation of PCR1 in cam, Colony 3 in 5ml LB+cam

From left to right: PCR8k,PCR8l,ladder (5000,2000,850,400,100bp)

Transformation of pSB1C3, BB1, BB6

New transformation of pSB1C3 from Spring distribution because the old colony was pink.

Transformation of BB1 and BB6.

BB1 plated on amp-Agar, BB6 and pSB1C3 plated on cam-Agar

-> Protocol: 19 Transformation (Sina Science Services)

Agarose gelelectrophoresis of PCR8 (long and short primer 13)

PCR8k=short primer 13, PCR8l=long primer 13

-> Protocol: 11 Agarose gel electrophoresis

result: PCR 8k and 8l shows right band with ~1200bp

PCR purification of PCR 8

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

result: ~70ng/µl; A260/A280=1.75

9-24-2010

New "over-day" culture of pSB1C3

Inoculate one culture from the ligation yesterday in 4ml LB+cam

Ligation of BB7₂

Mixture:

Inserts: each 5µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 7µl

Incubation: 5h 18°C; Inhibition: 20 min 65°C

->Protocols: 22 Ligation, 13 3A Method for Biobrick assembly

Colony PCRs of BB1 and BB6

2 Colonys were picked from each plate -> 4 charges

Mixture for 4 charges with 5µl:

PCR Mastermix 2x	10µl
Primer F	1.5µl
Primer R	1.5µl
H₂O	7µl
sum	20µl

+ Colony

from left to right: 1₁, 1₂, 6₁ and 6₂

-> Protocol: 21 PCR with Taq Mastermix

Annealing temperature: 53°C, standard protocol

Plasmid extraction of new PCR1 in cam vector

-> Protocol: 4 Plasmid extraction from cells

results:

concentration	82.5ng/µl
A260/A280	1.737

Agarose Gelelektrophoresis of 1₁, 1₂, 6₁ and 6₂

Plasmid extraction of pSB1C3 in cam vector

-> Protocol: 4 Plasmid extraction from cells

results:

concentration	22.5ng/µl
A260/A280	1.5

9-25-2010

weekend

9-26-2010

weekend

9-27-2010

Colony PCR of BB7₂, BB6 and BB1 plus a negativ charge as reference

PCR Mastermix	17.5µl
Primer forward	2.625µl
Primer revers	2.7µl
H₂O	12.1µl
sum	35µl = 5µl per charge

-> Protocol 24 Colony PCR

Ligation of BB10

CMV© E+S	5µl
PCR 8l X+P	5µl
ccdBamp	1µl
T4 Ligase-Buffer	2µl
T4 Ligase	0.5µl
H₂O	6.5µl
sum	20µl

-> Protocol 22 Ligation

ccdB	1µl
BB001/017/018/021	5µl
T4 Ligase-Buffer	2µl
T4 Ligase	0.5µl
H₂O	11.5µl
sum	20µl

-> Protocol 22 Ligation

Restriction digestion of PCR3₂, 8l, 10 and Primer 18+19 with E+P

	template	H₂O	BSA [1:10]	Buffer H	EcoRI	PstI
PCR3₂	10µl	5µl	2µl	2µl	0.5µl	0.5µl
PCR8l	10µl	5µl	2µl	2µl	0.5µl	0.5µl
Primer 18+19	2x7.5µl	--	2µl	2µl	0.5µl	0.5µl
PCR10	10µl	5µl	2µl	2µl	0.5µl	0.5µl

-> Protocol 5 Restriction digestion

9-28-2010

Plating BB017, BB021, BB018, BB001, BB7₂, BB10

Amp	BB7₂	BB10
Cam	BB001	BB017	BB018	BB021

Colony PCR BB1, BB6, BB7₂ with DreamTaq (each 2 colonies)

-> Protocol: 16 PCR with DreamTaq

PCR mixture for PCR7a

template	pick up colony from plate
primers*2	0.25µl*2
dNTPs	0.5µl
DreamTaq	0.05µl
10xbuffer	0.5µl
H₂O	3.45µl
sum	5µl

Primers for colony PCR: BBseqF, BBseqR

Annealing temperature: 53°C

Ligation of BB2, BB019, BB020, BB016, BB022

	BB2	BB019	BB020	BB016	BB022	Volume
PCR	5₂	8		3₂	10	each 5µl
PCR	6					each 5µl
Primer			18+19			each 5µl
ccdBamp	1µl
ccdBcam		1µl	1µl	1µl	1µl
T4 Ligase-Buffer	2µl	2µl	2µl	2µl	2µl
T4 Ligase	0.5µl	0.5µl	0.5µl	0.5µl	0.5µl
H₂O	6.5µl	11.4µl	11.4µl	11.4µl	11.4µl
sum	20µl	20µl	20µl	20µl	20µl

9-29-2010

plates BB7₂, BB017, BB10, BB021, BB001, BB018: nothing grown -> thrown into trash

colony-PCR of BB2/8, BB019, BB016, BB020, BB022 with Taq

->protocol: PCR with Taq

H₂O	6,7µl
Buffer 10x	1µl
MgCl₂	1µl
dNTPs	0,2µl
Primer forw. seq f	0,5µl
Primer rev. seq r	0,5µl
Taq	0,1µl

sum: 10µl; we made 100µl ->10 charges (2 colonies of each plate)

tube numbers:

1	2	3	4	5	6	7	8	9	10
BB2/BB8 - 1	BB2/BB8 - 2	BB016 - 1	BB016 - 2	BB019 - 1	BB019 - 2	BB020 - 1	BB020 - 2	BB022 - 1	BB022 - 2

PCR-program: taq53

agarose-gelelektrophoresis -> no bands visible

new plates made of BB1, BB4, BB6, BB7

Ligations BB7₂, BB017 (mit PCR5₁), BB10, BB021, BB001, BB018

	PCR (5 µl each)	PCR (5µl each)	ccdB(amp)	ccdB©	Buffer	T4 Ligase	H₂O
BB7₂O	1	3₂	1µl		2µl	0,5µl	6,5µl
BB017	5₁			1µl	2µl	0,5µl	11,5µl
BB10	CMV©	PCR8	1µl		2µl	0,5µl	6,5µl
BB021	9			1µl	2µl	0,5µl	11,5µl
BB001	1			1µl	2µl	0,5µl	11,5µl
BB018	6			1µl	2µl	0,5µl	11,5µl

restriction degestions

of PCR6, PCR5₂, PCR3₂, PCR1, ccdB(amp), ccdB(cam)

template	digestion with	volume
PCR 6	Xba1 & Pst1	I. 10µl II. 3µl
PCR5₂	EcoR1 & Pst1	20µl
PCR3₂	Xba1 & Pst1	10µl
PCR1	EcoR1 & Spe1	2µl
ccdB(amp)	EcoR1 & Pst1	20µl
cddB(cam)	EcoR1 & Pst1	20µl

->protocol: 5 Restriction digestion

colony-PCR of BB4/c3, BB6/c7, BB7₂/c5, BB1/c5, (BB11 as control) with Taq

->protocol: PCR with Taq

Mastermix:

H₂O	32µl
Buffer 10x	5µl
MgCl₂	5µl
dNTPs	1µl
Primer forw. seq f	2,5µl
Primer rev. seq r	2,5µl
Taq	0,5µl
DMSO	1,5µl
sum	50µl

->10µl for each charge

tube numbers:

1	2	3	4	5
BB4-c3	BB6-c7	BB7₂-c5	BB1-c5	BB11

PCR program:T_A = 53°C; elongation time=1min

9-30-2010

Colony PCR of BB2/BB8, BB6 K7&K8 and test PCR of BB11 with new dilutions and primers

Colony/BB11	2.5µl
H₂O	32µl
dNTP	1µl
Primer F	2.5µl
Primer R	2.5µl
MgCl₂	5µl
DMSO	1.5µl
Buffer 10x (with KCl)	5µl
Taq Polymerase	0.5µl
sum	56µl

-> Protocol 24 Colony PCR

PCR purification of the digestions of 6I, 6II, 5₂, 3₂, 1

	concentration [ng/µl]	A260/A280
6II	32.5	1.444
ccdBamp	0.0	---
ccdBcam	5.0	1.000
6I	0.0	---
5₂	20.0	1.143
3₂	27.5	1.57
1	25	1.667

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

Transformation of BB018, BB7₂, BB001, BB017, BB10 and BB021

-> Protocol 19 Transformation SSS

Plasmidextraction of the following overnight cultures: BB020(K1), BB018(K5), BB6(K5), BB022(K1), BB016(K1), BB019(K1), BB2(K1) and BB018(K3)

	concentration [ng/µl]	A260/A280
BB020(K1)	163	1.711
BB018(K5)	92.5	1.85
BB6(K5)	120	1.778
BB022(K1)	105	1.75
BB016(K1)	85	1.889
BB019(K1)	105	1.909
BB2(K1)	190	1.81
BB018	110	1.833

-> Protocol 4 Plasmid extraction from cells

10-01-2010

new test PCR of BB5 and BB11 with 20µl and 50µl volume

Master Mix

H₂O	67µl
dNTP	2µl
Primer F	5µl
Primer R	5µl
MgCl₂	10µl
Buffer 10x (with KCl)	10µl
Taq Polymerase	1µl
sum	100µl

->protocol: PCR with Taq

from left to right: ladder, BB5/50µl, BB5/20µl, BB11/20µl. BB5/50µl and BB11/20µl show the right band.

10-02-2010

weekend

10-03-2010

weekend

10-04-2010

sequencing

cycle, clean and run

	CMV (c)	PCR 1 (c)	BB4 ₇	BB4 ₈	BB018K5	BB018K3	BB6K5	BB2K1
template	1µl	2µl	3µl	3,8µl	2µl	2µl	2µl	1µl
primer (1:100)	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl
10mM TrispH 8,5	2,8 µl	1,8 µl	0,8 µl	0 µl	1,8 µl	1,8 µl	1,8 µl	1,8 µl
name of tube	028 FIR	001 F/R	4-7 F/R	4-8 F/R	018-5 F/R	018-3 F/R	6-5 F/R	2-1 F/R

PCR

	019	022	016	020
template (1:100 = 2ng)	2µl	2µl	2,5µl	1,5µl
Primer F	1µl	1µl	1µl	1µl
Primer R	1µl	1µl	1µl	1µl
dNTPs	0,4µl	0,4µl	0,4µl	0,4µl
Taq	0,2µl	0,2µl	0,2µl	0,2µl
Buffer	2µl	2µl	2µl	2µl
MgCl₂	2µl	2µl	2µl	2µl
H₂O	11,4µl	11,4µl	10,9µl	11,
colour of tube	orange	rose	green	blue

10-05-2010

agarose gel electrophoresis

schedule of the agarose gel electrophoresis:

gel 1	BB6K9	BB6K10	BB6K11	BBK12	BB6K13	BB4K5	ladder	BB4K6	BB4K7
	BB021K14	BB021K13	BB021K12	BB021K11	BB022K18	BB022K19	ladder	BB022K17	BB022K16
gel 2	BB022K12	BB022K11	CMV©K4	CMV©K5	BB001K5	BB001K6	BB001K7	BB001K8	ladder
	BB016K17	BB1K16	BB1K15	BB1K8	BB1K7	BB2_5₂K1	BB2_5₂K2	BB2_5₂K3	ladder
gel 3	BB017K14	BB017K13	BB017K12	BB017K11	BB018K17	BB018K16	BB018K15	BB018K14	ladder
	BB019K6	BB019K5	BB019K4	ladder	BB019K3	BB020K7	BB020K8	BB020K6	BB020K5

gel 1	BB4K8	BB4K9	BB4K10	BB021K10	BB021K15
	BB022K15	BB022K14	BB022K13	---	---
gel 2	BB001K9	BB001K10	BB016K11	BB016K12	BB016K13	BB016K14	BB016K15	BB016K16	---
	---	BB2_5₂K4	BB2_5₂K5	BB7₂K12	BB7₂K11	BB7₂K10	BB7₂K9	BB017K16	BB017K15
gel 3	BB018K13	BB018K12	BB018K11	BB018K10	BB018K9
	BB020K4	BB020K3	---	---

Gelbilder und Auswertung einfügen

overnight cultures of BB6K3, BB4K10, CMV©K4, BB001K5, BB001K9, BB016K11, BB1K8, BB2_5₂K3, BB7₂, BB017K14, BB018K10, BB019K6

10-06-2010

Plasmidextraction of the following overnight cultures: BB6(K13), BB4(K13), CMVc (K4), BB001(K5), BB001(K9), BB016(K11), BB1(K8), BB2(K3), BB7(K10), BB017(K14), BB018(K10) and BB019(K6)

	concentration [ng/µl]	A260/A280
BB6(K13)	265	1.8
BB4(K10)	40	2.0
CMVc(K4)	97.5	1.8
BB001(K5)	30	1.7
BB001(K9)	17.5	1.7
BB016(K11)	45	1.7
BB1(K8)	57.5	1.9
BB2(K3)	60	1.8
BB7(K10)	30	1.7
BB017(K14)	12.5	1.5
BB018(K10)	35	1.8
BB019(K6)	37.5	1.5

-> Protocol 4 Plasmid extraction from cells

10-07-2010

colony PCR of BB021, BB022 with Taq (53°C)

Primer fw	0.5 µl
Primer rev	0.5 µl
dNTPs	0.2 µl
Taq	0.1 µl
10xbuffer	1 µl
MgCl₂	1 µl
H₂O	6.7 µl

10-08-2010

plasmide extractions from 10-06-2010 were put in a lyophilizer and redissolved in less water (10-20 µl)

	concentration [ng/µl]	A260/A280
BB4(K10)	258	1,981
BB001(K5)	325	1.94
BB001(K9)	205	1.907
BB7(K10)	170	1.744
BB017(K14)	215	1.955
BB018(K10)	500	1.88
BB019(K6)	293	1.95

10-09-2010

Plasmid extraction BB018K5, BB018K6, BB021K17 and BB021K24

concentration of these samples to be measured on Monday

-> Protocol 4 Plasmid extraction from cells

10-10-2010

weekend

10-11-2010

Plasmid extraction BB018K5, BB018K6, BB021K24 and BB021K17 from 10-09-2010

	concentration [ng/µl]	A260/A280
BB018K5	55	1.571
BB018K6	92,5	1.762
BB021K24	27.5	1.833
BB021K17	37.5	1.508

Colony PCR BB01, BB018, BB6, BB252

stripe 1	BB001K5	K6	K7	K8	K9	K9	K10	BB252 K2
stripe 2	BB252 K3	K4	K5	BB6 K9	K10	K11	K12	K13
stripe 3	BB018 K9	K10	K11	K12	K13	K14	K15	K16
single sample	BB018 K17

gel picture colony PCR

foto einfügen!

Sequencing 8,9 and 10 (cycle, clean and run)

	BB001K9 for	BB001K9 rev	BB4K16 for	BB4K16 rev	BB001K5 for	BB001K5 rev	BB2_5₂K3	BB2_5₂K3
template	1µl	1µl	1µl	1µl	0,5µl	0,5µl	3,8µl	3,8µl
primer (1:100)	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl
10mM TrispH 8,5	2,8 µl	2,8 µl	2,8 µl	2,8 µl	3,3 µl	3,3 µl	0 µl	0 µl
name of tube	31-F	31-R	44-F	44-R	24-F	24-R	32-F	32-R

	BB7₂K10 for	BB7₂K10 rev	BB017K14 for	BB017K14 rev	BB018K10 for	BB018K10 rev	BB019K6 for	BB019K6 rev
template	1,5µl	1,5µl	1µl	1µl	0,5µl	0,5µl	0,8µl	0,8µl
primer (1:100)	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl
10mM TrispH 8,5	2,3 µl	2,3 µl	2,8 µl	2,8 µl	3,3 µl	3,3 µl	3 µl	3 µl
name of tube	14-F	14-R	21-F	21-R	13-F	13-R	34-F	34-R

	CMV for	CMV rev	BB018K5 for	BB018K5 rev	BB018K6 for	BB018K6 rev	BB021K17	BB021K17
template	2µl	2µl	3,8µl	3,8µl	2µl	2µl	3,8µl	3,8µl
primer (1:100)	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl	3,2 µl
10mM TrispH 8,5	1,8 µl	1,8 µl	0 µl	0 µl	1,8 µl	1,8 µl	0 µl	0 µl
name of tube	CMV-F	CMV-R	185-F	185-R	186-F	186-R	2117-F	2117-R

ligation BB020 BB022, Bb6 and BB5₂

-> Protocol 19 Transformation SSS

	BB020	BB022	BB6	BB5₂
insert (5µl)	18+19	PCR 10	CMV	PCR5<sub<2</sub>
insert (5µl)	-	-	BB5	PCR6
ccdB (1µl)	c	c	c	a
buffer	2µl	2µl	2µl	2µl
T4-ligase	0,5µl	0,5µl	0,5µl	0,5µl
H₂O	11,5µl	11,5µl	6,5µl	6,5µl

PCR to test self-made taq

template eGFP	1µl
Primer 20,21	each 1µl
buffer (10x)	2µl
MgCl	2µl
dNTPs	0,2µl
taq	0,3µl to 0,7µl (0,1µl steps)
H₂O	fill to 20 µl

anealing temperature 58°C, elongation time 30

@@ Line 1: / Line 1: @@
 __NOTOC__
 {{:Team:LMU-Munich/Templates/Page Header}}
-=Apoptosis Notebook=
+==<font color="#9933CC">'''ApoControl Notebook'''</font>==
-=='''Contents'''==
-{| class="wikitable centered" border="2" rules="rows" width="100%" style="border-color:white;"
+== '''What we did''' ==
+<b>Short description of our work, our results and our supporters</b>
+The creation of certain constructs was necessary for our two systems for cell selection by means of apoptosis: “Cut’N’Survive” and “Jump-Or-Die”. We searched for sources of the DNA sequences we needed and found several supporters which are listed below.
+Most genes and promoters were amplificated via PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. All PCRs worked out, even the fusion PCRs.
+The length of the PCR products were tested by agarose gel electrophoresis. We tried to sequence our PCR products, but obtained poor results and resorted to sequencing the plasmids.
+In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were digested with the appropriate restriction enzymes. The samples were cleaned with a PCR clean up kit or dephosphorylated to reduce false ligations.
+We ligated our constructs and several interim stages with the 3A-assembly according to our schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. Afterwards, some colonies were picked and we tested the insertion of the construct by colony PCR.
+If the colony PCR resulted in bands of the right size, we extracted the plasmids from overnight cultures and sequenced the samples with forward and reverse BioBrick primers.
+Unfortunately, not all BioBricks were cloned succesfully. However, we were able to produce 4 BioBricks, one of which represents a full construct while the other three are intermediates. The system wasn't completed on time, so we weren´t able to test them in eukarytic cell lines.
+<b>The protocols we used are listed here: </b> [[Team:LMU-Munich/Notebook#Protocols|Protocols]]
+<b>These Biobricks we submitted: </b>
+*BBa_K368004: attP+eGFP+SV40PA
+*BBa_K368011: eGFP+SV40PA
+*BBa_K368016: TEVrecognition site+N-degron+SF3b155
+*BBa_K368019: TEV-Protease+p14*+TEVrecognition site
+<b>Sources, helpers and supporters:</b>
+*	Prof. Dr. Angelika Böttger :
+**	prevTRE (tet-on CMV promoter; inducible by doxycycline in special cell lines)
+**	supported the construction ideas and would have given us the cells and mediums we would have needed
+**	SV40PA (Polyadenylation site): gave us a vector containing it
+**	Human Bak:  her assistant Erika Clement gave us appropriate cDNA
+*	Dr. Arnim Weber: submitted us a vector with human Bak
+*	Dr. Philipe Soriano: <html>
+<a href="http://www.ncbi.nlm.nih.gov/pubmed/17225864?dopt=Abstract"> (Raymond CS et al: High-Efficiency FLP and PhiC31 Site-Specific Recombination in Mammalian Cells (2007))</a>
+</html>
+**	Sequences of attB and attP site
+**	PhiC31o was bought via addgene
+*	Knop, M (Heidelberg): <html>
+<a href ="http://www.ncbi.nlm.nih.gov/pubmed?term=Efficient%20protein%20depletion%20by%20genetically%20controlled%20deprotection%20of%20a%20dormant%20N-degron">(Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009))</a>
+</html>
+**	TEVrecognition site+N-degron+SF3b155
+**	TEV-Protease+p14*+TEVrecognition site
+*	Prof. Dr. Thorsten Mascher:
+**	Helped with primer design, agarose gel electrophoresis apparatuses and trouble shooting
+*	Prof. Dr. Kirsten Jung:
+**	Helped with ideas and fundraising
+*	Dr. Susanne Gebhard:
+**	Helped with trouble-shooting and materials
+*	Prof. Dr. Andreas Brachmann:
+**	Sequenced our samples
+*	Partsregistry:
+**	eGFP (BBa_I714891)
+**	CMV-Promoter (BBa_J52034: this part was wrong: its lacI !!!)
+**	ccdB amp, cam, tet, kan in E.coli DH3
+=='''Contents'''>==
+{|class="wikitable centered" border="2" rules="rows" width="100%" style="border-color:white;"
 |-
 ! Week                       !! colspan="7" |Days
@@ Line 136: / Line 199: @@
 |}
-== 8-02-2010 ==
-* Some test text here.
-== 8-03-2010 ==
-'''Some test text in bold'''
-We created following tests:
-* test1
-* test2
-* test3
-- test4<br/>
-- test5
-== 8-04-2010 ==
-Example of a table
-{| class="wikitable" border="1"
-|-
-! 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:
-{|
-|H<sub>2</sub>O<sub>ddes</sub>
-|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 <br> test
-== 8-09-2010 ==
-text <font color="#FF0000">Knallroter Text</font><br>farbnummern für farbige schrift: http://html.nicole-wellinger.ch/hilfen/farbenverzeichnis.html
-<font color="#009933">test grüner text</font>
 == 8-10-2010 ==
@@ Line 250: / Line 240: @@
 -	3 ml LB-Media + 4 µl Kanamycin
--	Inoculated iangeimpft) with 1 colony of BBa_I714891 -> 37°C
+-	Inoculated with 1 colony of BBa_I714891 -> 37°C
@@ Line 330: / Line 320: @@
 - 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
+- <font color="#CC33CC">'''Correct DNA bands for 190-6 (~4800bp, ~1900bp, ~6700bp (undigested plasmid)) and eGFP (~2000bp (Plasmid), ~750 bp (Insert))'''</font>; CMV probably not digested (two bands; one probably normal, one supercoiled) and pDS7 not clear
 <font color="#009933">Restriction digest from CMV and pDS7</font>
@@ Line 346: / Line 336: @@
 - 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
 <font color="#009933">Plated CMV on Ampicllin-Agar</font>
@@ Line 534: / Line 524: @@
 - Agarose gel electrophoresis of the restriction digest of PhiC31o and PCR 1 and 6
-- the right bands found for PhiC31o (~2900,~2400,~250)
+- <font color="#CC33CC">'''the right bands found for PhiC31o (~2900,~2400,~250)'''</font>
-- the right band found for PCR1 (~450)
+- <font color="#CC33CC">'''the right band found for PCR1 (~450)'''</font>
 - no band found for PCR6; new electrophoresis needed with more DNA loaded
@@ Line 563: / Line 553: @@
 - new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown)
-- the right band found for PCR6 (~200)
+-<font color="#CC33CC"> '''the right band found for PCR6 (~200)'''</font>
 <font color="#009933">New overnight cultures of CMV and pDS7</font>
@@ Line 602: / Line 592: @@
 -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/11_Agarose_gel_electrophoresis|11 Agarorse gel electrophoresis]])
--> right DNA bands for pDS7 (~7000bp, ~1000bp)
+-> <font color="#CC33CC">'''right DNA bands for pDS7 (~7000bp, ~1000bp)'''</font>
 -> false DNA bands for CMV
@@ Line 621: / Line 611: @@
 |}
--> the right bands for PCR2a (~300bp) and PCR2b (~700bp)
+-><font color="#CC33CC"> '''the right bands for PCR2a (~300bp) and PCR2b (~700bp)'''</font>
 - New agarose gel electrophoresis with all of the PCR product for gel extraction (150V, 30min)
@@ Line 644: / Line 634: @@
 [[Image:GelverdauPCR3-1.jpg|400px|Gelfoto from PCR3]]
-left column: marker; most right column: PCR3
+left column: marker; rightmost column: PCR3
 -> Protocol:   [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] (150V, 30min)
@@ Line 764: / Line 754: @@
 <font color="#009933">PCR 2a gel extraction</font>
-- Quaigen kit (QuaiexII)
+- Quiagen kit (QuiaexII)
 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction| 14 QIAEX II gel extraction]]
@@ Line 778: / Line 768: @@
 [[Image:24_8_10_apo3.jpg|thumb|right|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
+- expected bands:<font color="#CC33CC"> '''right bands with 2ng and 5ng template (~700bp)'''</font>, no band with 10ng template
 <font color="#009933">CMV plasmid extraction</font>
@@ Line 1,084: / Line 1,074: @@
 |-
-|7b
+|<font color="#CC33CC">'''7b'''</font>
-|402bp
+|<font color="#CC33CC">'''402bp'''</font>
-|right band (~400bp)+ false band (~150bp)
+|<font color="#CC33CC">'''right band (~400bp)'''</font>+ false band (~150bp)
 |-
@@ Line 1,094: / Line 1,084: @@
 |-
-|10
+|<font color="#CC33CC">'''10'''</font>
-|1888bp
+|<font color="#CC33CC">'''1888bp'''</font>
-|right band (~1900bp)+false band (~500bp)
+|<font color="#CC33CC">'''right band (~1900bp)'''</font>+false band (~500bp)
 |-
@@ Line 1,194: / Line 1,184: @@
 - PCR7a, 9: false band at 200bp
-- ccdB: each digestion leads to a right band with ~ 650bp
+- <font color="#CC33CC">'''ccdB: each digestion leads to a right band with ~ 650bp'''</font>
 == 8-28-2010 ==
@@ Line 1,345: / Line 1,335: @@
 - PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu): no band shown
-- PCR3 (Phusion): right band (~1000bp)
+- <font color="#CC33CC">'''PCR3 (Phusion): right band (~1000bp)'''</font>
 <font color="#009933">New PCR  PCR4a, PCR4b, PCR7a, PCR9</font>
@@ Line 1,438: / Line 1,428: @@
 - results:
-- PCR3; right band (~1000bp) and side-product
+-<font color="#CC33CC"> '''PCR3; right band (~1000bp)'''</font> and side-product
 - PCR7a: no band
@@ Line 1,673: / Line 1,663: @@
 expected bands:
-*4a: 330bp -> P2 and P3 show right bands and "primer clouds"(?)
+*<font color="#CC33CC">'''4a: 330bp -> P2 and P3 show right bands '''</font>and "primer clouds"(?)
-*4b: 376bp -> P1, P2, P3 show right bands and "primer clouds" (?)
+*<font color="#CC33CC">'''4b: 376bp -> P1, P2, P3 show right bands'''</font> and "primer clouds" (?)
 == 9-02-2010 ==
@@ Line 1,808: / Line 1,798: @@
 from left to right: 4a*, 4a, 4b*, 4b, 7a Phusion, 7a Pfu, Ladder
--> result: 4b, 4b*: right bands (~330bp)
+-> <font color="#CC33CC">'''result: 4b, 4b*: right bands (~330bp)'''</font>
 -remain: false bands/no band
@@ Line 1,817: / Line 1,807: @@
 from left to right: ladder, 4 columns pathway, 4a gelextr., 4b gelextr.
--> results: slight right bands for 4a and 4b, no "primer clouds" anymore.
+-> <font color="#CC33CC">'''results: slight right bands for 4a and 4b'''</font>, no "primer clouds" anymore.
 == 9-03-2010 ==
@@ Line 2,150: / Line 2,140: @@
 !PCR nr.
 !1
-!2a
+!<font color="#CC33CC">2a</font>
-!2b
+!<font color="#CC33CC">2b</font>
-!3
+!<font color="#CC33CC">3</font>
-!4a
+!<font color="#CC33CC">4a</font>
-!7b
+!<font color="#CC33CC">7b</font>
 !9
-!10
+!<font color="#CC33CC">10</font>
 |-
 |expected band (bp)
@@ Line 2,170: / Line 2,160: @@
 |shown band(s)
 |550,200
-|300
+|<font color="#CC33CC">'''300'''</font>
-|750
+|<font color="#CC33CC">'''750'''</font>
-|1100
+|<font color="#CC33CC">'''1100'''</font>
-|300
+|<font color="#CC33CC">'''300'''</font>
-|450
+|<font color="#CC33CC">'''450'''</font>
 |900,1500
-|1900
+|<font color="#CC33CC">'''1900'''</font>
 |-
 |clean charge
 |
-|x
+|<font color="#CC33CC">'''x'''</font>
-|x
+|<font color="#CC33CC">'''x'''</font>
-|~x
+|<font color="#CC33CC">'''~x'''</font>
-|x
+|<font color="#CC33CC">'''x'''</font>
-|x
+|<font color="#CC33CC">'''x'''</font>
 |
-|x
+|<font color="#CC33CC">'''x'''</font>
 |-
 |}
@@ Line 2,476: / Line 2,466: @@
 [from left to right: PCR 1, 3, 4a, 5, 7a, 9, 10, Ladder]
-PCR3, 9, 10 with right bands.
+<font color="#CC33CC">'''PCR3, 9, 10 with right bands.'''</font>
 <font color="#009933">New PCR 1, 4a, 4b, 5, 7a with phusion</font>
@@ Line 2,532: / Line 2,522: @@
 [From left to right: Ladder, 1, 4a, 4b, 5, 7a]
-Only 4a has been amplified successfully.
+Only<font color="#CC33CC"> '''4a has been amplified successfully.'''</font>
@@ Line 2,539: / Line 2,529: @@
 [[Image: apo-10-9-10-2.jpg|400px|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
+from left to right:<font color="#CC33CC"> '''PCR 3, band ~700bp, PCR 4a, band ~300bp, PCR 5, bands ~550bp (5*), ~650bp (5), PCR 9, band ~800bp, PCR 10, band ~1900bp'''</font>
 results:
@@ Line 2,843: / Line 2,833: @@
 from left to right: 7a: 1:100 diluted: 48°C, 52°C, 56.1°C, undiluted: 48°C, 52°C, 56.1°C, 8 without Mutation
-weak right band (with fuzz)for 7a undiluated with best result for 56°C
+<font color="#CC33CC">'''weak right band (with fuzz)for 7a '''</font>undiluated with best result for 56°C
 -> <font color="#009933">new PCR 7a and "8"</font>
@@ Line 3,180: / Line 3,170: @@
 ::6-1 and 6-2: ?
-::6: ok
+::<font color="#CC33CC">'''6: ok'''</font>
 ::7a-1,7a-2,"8"-1,"8"-2: primerdimer-problem -> we ordered new primers!
@@ Line 3,316: / Line 3,306: @@
 From left to right: PCR1, PCR3, PCR5, Ladder, PCR6, PCR9, PCR10
--> right band for PCR 3,5,6,9,10; no band for PCR 1
+-> <font color="#CC33CC">'''right band for PCR 3,5,6,9,10'''</font>; no band for PCR 1
 [[Image:9 17 10apo2.jpg| 400px |9 17 10 Gelphoto2]]
@@ Line 3,486: / Line 3,476: @@
 <font color="#009933">Colony PCR of Ligations 3, 5<sub>1</sub>, 5<sub>2</sub>, 6, 9, 10 and PCR of CMV1 (to test if right)</font>
-1-4, 9 1-4, 6 1-4, 5<sub>1</sub> 1-4, 5<sub>2</sub> 1-4, 3 1-4 Colonies pickt and put in following Mix:
+1-4, 9 1-4, 6 1-4, 5<sub>1</sub> 1-4, 5<sub>2</sub> 1-4, 3 1-4 Colonies picked and put in following Mix:
 {|
@@ Line 3,533: / Line 3,523: @@
 Below: From left to right: 6.1, 6.2, 6.3, 6.4, 9.1, 9.2, 9.3, 9.4, Ladder, 10.1, 10.2, 10.3, 10.4, CMV1
--> right bands for 6.3 and 6.4, CMV1 ~1200bp (we think that this is right, as Biobrick sequenzing information indicates that it isn't 654bp but about 1200bp)
+-> <font color="#CC33CC">'''right bands for 6.3 and 6.4, CMV1 ~1200bp (we think that this is right, as Biobrick sequenzing information indicates that it isn't 654bp but about 1200bp)'''</font>
@@ Line 3,650: / Line 3,640: @@
-verified products:
+<font color="#CC33CC">'''verified products:7a (~800bp); JD.2.1; JD.2.2(each ~1000bp);JD.3<sub>1</sub>.1(~2000bp); CS.1a.2(~1500bp)'''</font>
-a (~800bp); JD.2.1; JD.2.2(each ~1000bp);JD.3<sub>1</sub>.1(~2000bp); CS.1a.2(~1500bp)
 no bands shown: CS.2b.1, CS.2b.2, PCR1.1, PCR1.CMV,3.8, 5<sub>1</sub>.7, 5<sub>2</sub>.6, 9.7, 10.5, 10.6
@@ Line 3,832: / Line 3,821: @@
 From left to right: PCR7b, pathway (3x),ladder (5000,2000,850,400,100bp)
-result: PCR 7b shows right band with ~400bp
+result: <font color="#CC33CC">'''PCR 7b shows right band with ~400bp '''</font>
 <font color="#009933">PCR clean up of PCR 7b</font>
@@ Line 3,990: / Line 3,979: @@
 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]]
-result: PCR 8k and 8l shows right band with ~1200bp
+result:<font color="#CC33CC"> '''PCR 8k and 8l shows right band with ~1200bp'''</font>
 <font color="#009933">PCR purification of PCR 8</font>
@@ Line 4,038: / Line 4,027: @@
 + Colony
+[[Image: apo-24.9.10.jpg|200px|thumb|right|from left to right: 1<sub>1</sub>, 1<sub>2</sub>, 6<sub>1</sub> and 6<sub>2</sub>]]
 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]]
@@ Line 4,046: / Line 4,037: @@
 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
-[[Image: apo-24.9.10.jpg|200px|thumb|right|from left to right: 1<sub>1</sub>, 1<sub>2</sub>, 6<sub>1</sub> and 6<sub>2</sub>]]
 results:
@@ Line 4,377: / Line 4,366: @@
 tube numbers:
-{|
+{| class="wikitable" border="1"
 |-
 |1
@@ Line 4,410: / Line 4,399: @@
 <font color="#009933">Ligations BB7<sub>2</sub>, BB017 (mit PCR5<sub>1</sub>), BB10, BB021, BB001, BB018</font>
-{|
+{| class="wikitable" border="1"
 |-
 |
@@ Line 4,480: / Line 4,469: @@
 of PCR6, PCR5<sub>2</sub>, PCR3<sub>2</sub>, PCR1, ccdB(amp), ccdB(cam)
-{|
+{| class="wikitable" border="1"
 |-
 |template
@@ Line 4,555: / Line 4,544: @@
 tube numbers:
-{|
+{| class="wikitable" border="1"
 |-
 |1
@@ Line 4,726: / Line 4,715: @@
 template: BB5/50µl: 5ng, 20µl: 1.5ng; BB11/20µl: 3ng
--> Protocol [[Team:LMU-Munich/Notebook/Protocols/24 Colony PCR|24 Colony PCR]]
+->protocol: [[Team:LMU-Munich/Notebook/Protocols/25 PCR with Taq|PCR with Taq]]
+[[Image:01.10.10. apo.jpg|01.10.10. Gel photo]]
+from left to right: ladder, BB5/50µl, BB5/20µl, BB11/20µl. BB5/50µl and BB11/20µl show the right band.
 == 10-02-2010 ==
@@ Line 4,734: / Line 4,727: @@
 == 10-04-2010 ==
-text
+sequencing
+cycle, clean and run
+{| class="wikitable" border="1"
+|-
+!
+! CMV (c)
+! PCR 1 (c)
+! BB4 <sub>7</sub>
+! BB4 <sub>8</sub>
+! BB018K5
+! BB018K3
+! BB6K5
+! BB2K1
+|-
+| template
+| 1µl
+| 2µl
+| 3µl
+| 3,8µl
+| 2µl
+| 2µl
+| 2µl
+| 1µl
+|-
+| primer (1:100)
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+|-
+| 10mM TrispH 8,5
+| 2,8 µl
+| 1,8 µl
+| 0,8 µl
+| 0 µl
+| 1,8 µl
+| 1,8 µl
+| 1,8 µl
+| 1,8 µl
+|-
+|name of tube
+| 028 FIR
+| 001 F/R
+| 4-7 F/R
+| 4-8 F/R
+| 018-5 F/R
+| 018-3 F/R
+| 6-5 F/R
+| 2-1 F/R
+|}
+PCR
+{| class="wikitable" border="1"
+|-
+!
+! 019
+! 022
+! 016
+! 020
+|-
+| template (1:100 = 2ng)
+| 2µl
+| 2µl
+| 2,5µl
+| 1,5µl
+|-
+| Primer F
+| 1µl
+| 1µl
+| 1µl
+| 1µl
+|-
+| Primer R
+| 1µl
+| 1µl
+| 1µl
+| 1µl
+|-
+| dNTPs
+| 0,4µl
+| 0,4µl
+| 0,4µl
+| 0,4µl
+|-
+| Taq
+| 0,2µl
+| 0,2µl
+| 0,2µl
+| 0,2µl
+|-
+| Buffer
+| 2µl
+| 2µl
+| 2µl
+| 2µl
+|-
+|MgCl<sub>2</sub>
+| 2µl
+| 2µl
+| 2µl
+| 2µl
+|-
+|H<sub>2</sub>O
+| 11,4µl
+| 11,4µl
+| 10,9µl
+| 11,
+|-
+|colour of tube
+| orange
+| rose
+| green
+| blue
+|}
 == 10-05-2010 ==
-text
+<font color="#009933">Colony PCRs of BB6, BB4, BB021, BB022, CMV©, BB001, BB016, BB1, BB2<sub>5<sub>2</sub></sub>, BB7<sub>2</sub>, BB017, BB018, BB019, BB020</font>
+<font color="#009933">agarose gel electrophoresis</font>
+schedule of the agarose gel electrophoresis:
+{| class="wikitable" border="1"
+| gel 1
+| BB6K9
+| BB6K10
+| BB6K11
+| BBK12
+| BB6K13
+| BB4K5
+| ladder
+| BB4K6
+| BB4K7
+|-
+|
+| BB021K14
+| BB021K13
+| BB021K12
+| BB021K11
+| BB022K18
+| BB022K19
+| ladder
+| BB022K17
+| BB022K16
+|-
+| gel 2
+| BB022K12
+| BB022K11
+| CMV©K4
+| CMV©K5
+| BB001K5
+| BB001K6
+| BB001K7
+| BB001K8
+| ladder
+|-
+|
+| BB016K17
+| BB1K16
+| BB1K15
+| BB1K8
+| BB1K7
+| BB2<sub>5<sub>2</sub></sub>K1
+| BB2<sub>5<sub>2</sub></sub>K2
+| BB2<sub>5<sub>2</sub></sub>K3
+| ladder
+|-
+| gel 3
+| BB017K14
+| BB017K13
+| BB017K12
+| BB017K11
+| BB018K17
+| BB018K16
+| BB018K15
+| BB018K14
+| ladder
+|-
+|
+| BB019K6
+| BB019K5
+| BB019K4
+| ladder
+| BB019K3
+| BB020K7
+| BB020K8
+| BB020K6
+| BB020K5
+|}
+{| class="wikitable" border="1"
+| gel 1
+| BB4K8
+| BB4K9
+| BB4K10
+| BB021K10
+| BB021K15
+|
+|
+|
+|
+|-
+|
+| BB022K15
+| BB022K14
+| BB022K13
+| ---
+| ---
+|
+|
+|
+|
+|-
+| gel 2
+| BB001K9
+| BB001K10
+| BB016K11
+| BB016K12
+| BB016K13
+| BB016K14
+| BB016K15
+| BB016K16
+| ---
+|-
+|
+| ---
+| BB2<sub>5<sub>2</sub></sub>K4
+| BB2<sub>5<sub>2</sub></sub>K5
+| BB7<sub>2</sub>K12
+| BB7<sub>2</sub>K11
+| BB7<sub>2</sub>K10
+| BB7<sub>2</sub>K9
+| BB017K16
+| BB017K15
+|-
+|gel 3
+| BB018K13
+| BB018K12
+| BB018K11
+| BB018K10
+| BB018K9
+|
+|
+|
+|
+|-
+|
+| BB020K4
+| BB020K3
+| ---
+| ---
+|
+|
+|
+|
+|
+|}
+Gelbilder und Auswertung einfügen
+[[Image:5.10.10apo1,2,3.jpg|400px|9 15 10 Gelphoto]]
+<font color="#009933">overnight cultures of BB6K3, BB4K10, CMV©K4, BB001K5, BB001K9, BB016K11, BB1K8, BB2<sub>5<sub>2</sub></sub>K3, BB7<sub>2</sub>, BB017K14, BB018K10, BB019K6</font>
 == 10-06-2010 ==
-text
+<font color="#009933">Plasmidextraction of the following overnight cultures: BB6(K13), BB4(K13), CMVc (K4), BB001(K5), BB001(K9), BB016(K11), BB1(K8), BB2(K3), BB7(K10), BB017(K14), BB018(K10) and BB019(K6) </font>
+{|
+|
+| concentration [ng/µl]
+| A260/A280
+|-
+| BB6(K13)
+| 265
+| 1.8
+|-
+| BB4(K10)
+| 40
+| 2.0
+|-
+| CMVc(K4)
+| 97.5
+| 1.8
+|-
+| BB001(K5)
+| 30
+| 1.7
+|-
+| BB001(K9)
+| 17.5
+| 1.7
+|-
+| BB016(K11)
+| 45
+| 1.7
+|-
+| BB1(K8)
+| 57.5
+| 1.9
+|-
+| BB2(K3)
+| 60
+| 1.8
+|-
+| BB7(K10)
+| 30
+| 1.7
+|-
+| BB017(K14)
+| 12.5
+| 1.5
+|-
+| BB018(K10)
+| 35
+| 1.8
+|-
+|BB019(K6)
+| 37.5
+| 1.5
+|-
+|}
+-> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 == 10-07-2010 ==
-text
+<font color="#009933"> colony PCR of BB021, BB022 with Taq (53°C) </font>
+{|
+| Primer fw
+| 0.5 µl
+|-
+| Primer rev
+| 0.5 µl
+|-
+| dNTPs
+| 0.2 µl
+|-
+| Taq
+| 0.1 µl
+|-
+| 10xbuffer
+| 1 µl
+|-
+| MgCl<sub>2</sub>
+| 1 µl
+|-
+| H<sub>2</sub>O
+| 6.7 µl
+|-
+|}
 == 10-08-2010 ==
-text
+<font color="#009933">
+plasmide extractions from 10-06-2010 were put in a lyophilizer and redissolved in less water (10-20 µl)
+</font>
+{|
+|
+| concentration [ng/µl]
+| A260/A280
+|-
+| BB4(K10)
+|     258
+|     1,981
+|-
+| BB001(K5)
+|     325
+|     1.94
+|-
+| BB001(K9)
+|     205
+|     1.907
+|-
+| BB7(K10)
+|     170
+|     1.744
+|-
+| BB017(K14)
+|     215
+|     1.955
+|-
+| BB018(K10)
+|     500
+|     1.88
+|-
+|BB019(K6)
+|     293
+|     1.95
+|-
+|}
 == 10-09-2010 ==
-weekend
+<font color="#009933">Plasmid extraction BB018K5, BB018K6, BB021K17 and BB021K24</font color>
+concentration of these samples to be measured on Monday
+-> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]]
 == 10-10-2010 ==
 weekend
 == 10-11-2010 ==
-text
-== 10-12-2010 ==
-text
-== 10-13-2010 ==
-text
-== 10-14-2010 ==
-text
-== 10-15-2010 ==
-text
-== 10-16-2010 ==
-weekend
-== 10-17-2010 ==
-weekend
-== 10-18-2010 ==
-text
-== 10-19-2010 ==
-text
-== 10-20-2010 ==
-text
-== 10-21-2010 ==
-text
-== 10-22-2010 ==
-text
-== 10-23-2010 ==
-weekend
-== 10-24-2010 ==
-weekend
-== 10-25-2010 ==
+<font color="#009933">Plasmid extraction BB018K5, BB018K6, BB021K24 and BB021K17 from 10-09-2010
-text
+</font>
-== 10-26-2010 ==
-text
-== 10-27-2010 ==
+{|
-text
+|
-== 10-28-2010 ==
+| concentration [ng/µl]
-text
+| A260/A280
-== 10-29-2010 ==
+|-
-text
+| BB018K5
-== 10-30-2010 ==
+|     55
-weekend
+|     1.571
-== 10-31-2010 ==
+|-
-weekend
+| BB018K6
+|     92,5
+|     1.762
+|-
+| BB021K24
+|     27.5
+|     1.833
+|-
+| BB021K17
+|     37.5
+|     1.508
+|-
+|}
+<font color="#009933">Colony PCR BB01, BB018, BB6, BB252</font>
+{|
+| stripe 1
+| BB001K5
+| K6
+| K7
+| K8
+| K9
+| K9
+| K10
+| BB252 K2
+|-
+| stripe 2
+| BB252 K3
+| K4
+| K5
+| BB6 K9
+| K10
+| K11
+| K12
+| K13
+|-
+| stripe 3
+| BB018 K9
+| K10
+| K11
+| K12
+| K13
+| K14
+| K15
+| K16
+|-
+|single sample
+|BB018 K17
+|-
+|}
+gel picture colony PCR
+foto einfügen!
+<font color="#009933">Sequencing 8,9 and 10 (cycle, clean and run)</font>
+{| class="wikitable" border="1"
+|-
+!
+! BB001K9 for
+! BB001K9 rev
+! BB4K16 for
+! BB4K16 rev
+! BB001K5 for
+! BB001K5 rev
+! BB2<sub>5<sub>2</sub></sub>K3
+! BB2<sub>5<sub>2</sub></sub>K3
+|-
+| template
+| 1µl
+| 1µl
+| 1µl
+| 1µl
+| 0,5µl
+| 0,5µl
+| 3,8µl
+| 3,8µl
+|-
+| primer (1:100)
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+|-
+| 10mM TrispH 8,5
+| 2,8 µl
+| 2,8 µl
+| 2,8 µl
+| 2,8 µl
+| 3,3 µl
+| 3,3 µl
+| 0 µl
+| 0 µl
+|-
+|name of tube
+| 31-F
+| 31-R
+| 44-F
+| 44-R
+| 24-F
+| 24-R
+| 32-F
+| 32-R
+|}
+{| class="wikitable" border="1"
+|-
+!
+! BB7<sub>2</sub>K10 for
+! BB7<sub>2</sub>K10 rev
+! BB017K14 for
+! BB017K14 rev
+! BB018K10 for
+! BB018K10 rev
+! BB019K6 for
+! BB019K6 rev
+|-
+| template
+| 1,5µl
+| 1,5µl
+| 1µl
+| 1µl
+| 0,5µl
+| 0,5µl
+| 0,8µl
+| 0,8µl
+|-
+| primer (1:100)
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+|-
+| 10mM TrispH 8,5
+| 2,3 µl
+| 2,3 µl
+| 2,8 µl
+| 2,8 µl
+| 3,3 µl
+| 3,3 µl
+| 3 µl
+| 3 µl
+|-
+|name of tube
+| 14-F
+| 14-R
+| 21-F
+| 21-R
+| 13-F
+| 13-R
+| 34-F
+| 34-R
+|}
+{| class="wikitable" border="1"
+|-
+!
+! CMV for
+! CMV rev
+! BB018K5 for
+! BB018K5 rev
+! BB018K6 for
+! BB018K6 rev
+! BB021K17
+! BB021K17
+|-
+| template
+| 2µl
+| 2µl
+| 3,8µl
+| 3,8µl
+| 2µl
+| 2µl
+| 3,8µl
+| 3,8µl
+|-
+| primer (1:100)
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+| 3,2 µl
+|-
+| 10mM TrispH 8,5
+| 1,8 µl
+| 1,8 µl
+| 0 µl
+| 0 µl
+| 1,8 µl
+| 1,8 µl
+| 0 µl
+| 0 µl
+|-
+|name of tube
+| CMV-F
+| CMV-R
+| 185-F
+| 185-R
+| 186-F
+| 186-R
+| 2117-F
+| 2117-R
+|}
+<font color="#009933">ligation BB020 BB022, Bb6 and BB5<sub>2</sub></font color>
+-> Protocol [[Team:LMU-Munich/Notebook/Protocols/19 Transformation SSS|19 Transformation SSS]]
+{| class="wikitable" border="1"
+|-
+!
+! BB020
+! BB022
+! BB6
+! BB5<sub>2</sub>
+|-
+| insert (5µl)
+| 18+19
+| PCR 10
+| CMV
+| PCR5<sub<2</sub>
+|-
+| insert (5µl)
+| -
+| -
+| BB5
+| PCR6
+|-
+| ccdB (1µl)
+| c
+| c
+| c
+| a
+|-
+| buffer
+| 2µl
+| 2µl
+| 2µl
+| 2µl
+|-
+| T4-ligase
+| 0,5µl
+| 0,5µl
+| 0,5µl
+| 0,5µl
+|-
+| H<sub>2</sub>O
+| 11,5µl
+| 11,5µl
+| 6,5µl
+| 6,5µl
+|}
+<font color="#009933">PCR to test self-made taq</font color>
+{|
+| template eGFP
+| 1µl
+|-
+| Primer 20,21
+| each 1µl
+|-
+| buffer (10x)
+| 2µl
+|-
+| MgCl
+| 2µl
+|-
+| dNTPs
+| 0,2µl
+|-
+| taq
+| 0,3µl to 0,7µl (0,1µl steps)
+|-
+| H<sub>2</sub>O
+| fill to 20 µl
+|-
+|}
+anealing temperature 58°C, elongation time 30''
 {{:Team:LMU-Munich/Templates/Page Footer}}