Team:ATOMS-Turkiye/Extras/Notebook

• Tour
• Project
• Results
• P&P
• Team
• Extras

• Problems
• Ulcer
• Cancer Switch
• Modeling
• Safety
• Future Plan

• ATOMs
• Attributions
• Collaboration

• Notebook
• Protocols
• Achievements
• Official Part Page
• Interlab Study

• Virtual Hospital
• Video
• SynBio Day
• Outreach
• Street interview
• Ethics

Team ATOMS-Turkiye

atoms2015@googlegroups.com

NOTEBOOK

December

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

January

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

February

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

March

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

April

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

May

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

June

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

Week 1, 29.06.-30.06.

Gradient PCR of „pCAG with CMV-Enhancer FWD/Cβ-Actin REV. Primers” (29.06.2015)

Gradient PCR from pCAGGS
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	16.3 ul	2.0 ul	25.0 ul
9X	22.5 ul	22.5 ul	4.5 ul	4.5 ul	4.5 ul	1.8 ul	146.7 ul	18.0 ul	225.0 ul

57-64˚C

Results weren’t matching with expected results, experiment will be repeated.

GEL GÖRÜNTÜSÜ

(30.06.2015)

Gradient PCR from pCAGGS
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	16.3 ul	2.0 ul	25.0 ul
9X	22.5 ul	22.5 ul	4.5 ul	4.5 ul	4.5 ul	1.8 ul	146.7 ul	18.0 ul	225.0 ul

52-59˚C

Results weren’t matching with expected results, experiment will be repeated.

GEL GÖRÜNTÜSÜ

July

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

Week 1, 01.07.-05.07.

Gradient PCR of „pCAG with CAG FWD/CAG REV. Primers/pCMV REV.” (01.07.2015)

Gradient PCR from pCAGGS (CAG FWD – CAG REVERSE)
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	16.3 ul	2.0 ul	25.0 ul
9X	22.5 ul	22.5 ul	4.5 ul	4.5 ul	4.5 ul	1.8 ul	146.7 ul	18.0 ul	225.0 ul

60-68˚C

Results weren’t matching with expected results, experiment will be repeated.

GEL GÖRÜNTÜSÜ

Gradient PCR from pCAGGS (CAG FWD – Chicken β Aktin REVERSE)
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	16.3 ul	2.0 ul	25.0 ul
9X	22.5 ul	22.5 ul	4.5 ul	4.5 ul	4.5 ul	1.8 ul	146.7 ul	18.0 ul	225.0 ul

60-68˚C

Results weren’t matching with expected results, experiment will be repeated.

GEL GÖRÜNTÜSÜ

Protocols of „Phusion Pol, and Q5 Polymerase” (02.07.2015)

Phusion DNA Polymerase
	dNTP	Fwd primer	Rev primer	Buffer	Phusion Pol.	ddH₂O	DNA	Total
1x	0.4 ul	2.0 ul	2.0 ul	4.0 ul	0.2 ul	10.4 ul	1.0 ul	20.0 ul

Cycling
	98˚C	98˚C	???˚C	72˚C	72˚C	Cycle
Time	2’	10’’	30’’	1’	5’	???x

Q5 DNA Polymerase
	dNTP	Fwd primer	Rev primer	Buffer	Q5 Pol.	ddH₂O	DNA	Total
1x	0.5 ul	2.5 ul	2.5 ul	5.0 ul	0.25 ul	8.25 ul	1.0 ul	20.0 ul

Cycling
	98˚C	98˚C	???˚C	72˚C	72˚C	Cycle
Time	2’	10’’	30’’	1’	5’	???x

Defterde jel görüntüsü yok.

Week 2, 05.07.-12.07.

Gradient PCR of „pCAG with CAG-FWD/CAG REV. Primers and Phusion Pol.” (07.07.2015)

Gradient PCR from pCAGGS
	dNTP	Fwd primer	Rev primer	?????
Buffer	Phusion Pol.	ddH₂O	DNA	Total
1x	0.4 ul	2.0 ul	2.0 ul	4.0 ul	0.2 ul	10.4 ul	1.0 ul	20.0 ul

62-64˚C

Result: Gel extraction was performed. PCR (+): 680 bp

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	eb	biospec	7/8/2015 1:56:43 AM	0.1	ng/ul	0.002	-0.009	-0.25	-0.19	DNA	50.00
2	pCAG	biospec	7/8/2015 1:58:14 AM	13.7	ng/ul	0.275	0.138	1.98	0.15	DNA	50.00

Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” (09.07.2015)

Digestion of “pTRE” and “Promoter pCAG”
	pTRE (1536 ng/ul)	pCAG (promoter) (13.7 ng/ul)	EcoRI	XhoI	Neb 3.1 Buffer	ddH₂O	Total
1	3.2 ul	-	0.5 ul	0.5 ul	2.0 ul	13.8 ul	20.0 ul
2	-	10.0 ul	0.5 ul	0.5 ul	2.0 ul	7.0 ul	20.0 ul

pTRE-delta-TRE was made after digestion.

Concentration: 99.9 ng/ul

The final concentration of pCAG is 6.855 ng/ul.

Ligation of „pTRE TRE“ and „Digested promoter pCAG“
	pTRE TRE
pCAG	T4 DNA Ligase	Buffer	ddH₂O	Total
1:1	3.0 ul	7.0 ul	0.5 ul	2.0 ul	7.5 ul	20.0 ul

Ligation products were transformed into E.Coli/BL321 strain.

Result: No colonies were observed.

Digestion of “pTEToff and pET45 Vectors” (10.07.2015)

Digestion of “pTEToff and pET45 Vectors”
	pTEToff (1141 ng/ul)	pET45 (485 ng/ul)	XhoI (Neb)	BamHI (Neb)	SalI (Thermo)	HindIII (Thermo)	Cut Smart Buffer	Fast Digest Buffer	ddH₂O	Total
1	3.1 ul	-	-	-	0.5 ul	0.5 ul	-	2.0 ul	13.9 ul	20.0 ul	37˚C 1h
2	-	4.1 ul	0.5 ul	0.5 ul	-	-	2.0 ul	-	12.9 ul	20.0 ul	37˚C 2h

Result: Bands were at the expected section. Gel extraction was made.

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	eb	biospec	7/10/2015 11:37:54 PM	-0.4	ng/ul	-0.008	-0.015	0.58	-0.39	DNA	50.00
2	pET45 x+b	biospec	7/10/2015 11:40:59 PM	59.0	ng/ul	1.180	0.612	1.93	0.74	DNA	50.00
3	pTEToff s+h	biospec	7/10/2015 11:41:58 PM	55.5	ng/ul	1.110	0.581	1.91	0.25	DNA	50.00

Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” – Repeat (08.07.2015)

Digestion of “pTRE with EcoRI/XhoI”
	pTRE	XhoI	EcoRI	Neb 2.1 Buffer	ddH₂O	Total
1	3.2 ul	0.5 ul	0.5 ul	2.0 ul	13,8 ul	20.0 ul	37˚C 2h
2	3.2 ul	0.5 ul	0.5 ul	2.0 ul	13,8 ul	20.0 ul	37˚C 2h/0.5 ul CIP/37˚C 30’/50˚C 30’

Result: Gel extraction was made.

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	eb	biospec	7/9/2015 6:32:17 PM	-0.7	ng/ul	-0.015	-0.022	0.66	0.21	DNA	50.00
2	hre cmv mini	biospec	7/9/2015 6:34:10 PM	13.8	ng/ul	0.277	0.141	1.97	0.03	DNA	50.00
3	ptre delta tre cip -	biospec	7/9/2015 6:34:54 PM	88.7	ng/ul	1.774	0.941	1.88	0.24	DNA	50.00
4	ptre delta tre cip +	biospec	7/9/2015 6:35:35 PM	86.0	ng/ul	1.721	0.947	1.82	0.25	DNA	50.00

Creating “Plasmid pCAG” – Continue (12.07.2015)

Ligation of „pTRE TRE“ and „Digested promoter pCAG“
	pTRE TRE CIP (+)	pTRE TRE CIP (-)	pCAG (6.85 ng/ul)	T4 DNA Ligase	Buffer	ddH₂O	Total
1	3.0 ul	-	7.0 ul	0.5 ul	2.0 ul	7.5 ul	20.0 ul
2	-	3.0 ul	7.0 ul	0.5 ul	2.0 ul	7.5 ul	20.0 ul

Room Temperature 1h

Sonuc: Transformation was made. No colonies were observed at first plate. At the second plate there was five colonies. Colony PCR will be made.

Week 3, 13.07.-19.07.

Creating “Plasmid pCAG” – Continue (13.07.2015)

Colony PCR from “pCAG” with “pTRE Luc fwd/SV40 polyA re. primers”
	MgCl₂	(NH₄)2SO₄	pTRE Luc fwd	SV40 rev	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
6X	15.0 ul	15.0 ul	6.0 ul	6.0 ul	3.0 ul	1.2 ul	73.8 ul		120.0 ul

Cycling
	95˚C	95˚C	55˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated.

(+) bant: 923 bp

(-) bant: 705 bp

GEL GÖRÜNTÜSÜ

Creating “Plasmid pCAG” – Repeat (19.07.2015)

Ligation of „pTRE TRE“ and „Digested Promoter pCAG“
	pTRE TRE CIP (+)	pTRE TRE CIP (-)	pCAG (6.85 ng/ul)	T4 DNA Ligase	Buffer	ddH₂O	Total
1	3.0 ul	-	2.5 ul	0.5 ul	2.0 ul	12.0 ul	20.0 ul
2	-	3.0 ul	2.5 ul	0.5 ul	2.0 ul	12.0 ul	20.0 ul

Vector:Insert

3:1

RT 2h

Transformation at BL21.

CIP (+): No colonies were absorved.

CIP (-): Colony PCR will be made.

Week 4, 20.07.-26.07.-31.07.

Creating “Plasmid pCAG” – Continue (20.07.2015)

Colony PCR from “pCAG” with “pTRE Luc fwd/SV40 polyA re. primers”
	MgCl₂	(NH₄)2SO₄	pTRE Luc fwd	SV40 rev	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
6X	15.0 ul	15.0 ul	6.0 ul	6.0 ul	3.0 ul	1.2 ul	73.8 ul		120.0 ul

Cycling
	95˚C	95˚C	55˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated.

(+) bant: 923 bp

(-) bant: 705 bp

GEL GÖRÜNTÜSÜ

Creating “pCAG (Plasmid) – Repeat (23.07.2015)

PCR from “pCAGGS”
	dNTP	CAG fwd	CAG rev	Chicken β Akt. Rev	pCAGGS	Phusion Pol	6C??
GC??
Buffer	ddH₂O	Total
1	2.0 ul	10.0 ul	10.0 ul	-	5.0 ul	1.0 ul	20.0 ul	52.0 ul	100.0 ul
2	2.0 ul	10.0 ul	-	10.0 ul	5.0 ul	1.0 ul	20.0 ul	52.0 ul	100.0 ul

Cycling
	98˚C	98˚C	64/68˚C	72˚C	72˚C	Cycle
Time	2’	10’’	30’’	1’	5’	35x

Gel Extraction will made.

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	eb	biospec	7/23/2015 6:56:38 PM	0.2	ng/ul	0.005	0.005	0.92	0.14	DNA	50.00
2	pcag e	biospec	7/23/2015 6:58:03 PM	42.9	ng/ul	0.858	0.450	1.91	1.97	DNA	50.00
3	pcag y	biospec	7/23/2015 6:58:53 PM	23.3	ng/ul	0.466	0.233	2.00	1.94	DNA	50.00

Resuspension of “Newly Arrived G-Blocks from IDT” (S. 23/23.7.2015)

100 ul TE for all tubes.

		ng	fmol	TE ul	fmol/ul	ul of inserts for 75 fmol
1	Toehold for cola	1000	1523	100	15.23	4.92449
2	TnrA-pTnrA-RFP	1000	1032	100	10.32	7.26744
3	ColA-KanR-dTer	1000	816	100	8.16	9.19118
4	HNS for PET	500	1578	100	15.78	4.75285
5	HNS-T108I for PET	500	1578	100	15.78	4.75285
6	potB59-pomA for PET	1000	892	100	8.92	8.40807
7	Gad E – for PET	500	1291	100	12.91	5.80945
8	TlpB for PET	1000	901	100	9.01	8.32408
9	DAMP-Pex for PET/pcolA	1000	2089	100	20.89	3.59023
10	Tev Protease for PET	1000	1948	100	19.48	3.8501
11	miRNA switch- miR373- BS for pTET	500	2212	100	22.12	3.3906
12	LacO- DsRed- miR26a-375 pC	1000	1709	100	17.09	4.38853
13	mLacI-miR373 BS for pTRE	1000	1280	100	12.8	5.85938
14	miRNA switch- miR 21 BS- miR 223	1000	1902	100	19.02	3.94322
15	mLacI-miR223 BS miR 21 BS for pTRE	1000	1272	100	12.72	5.89623
16	Trigger RNA for pSB1C3	250	1910	100	19.1	3.9267
17	PsicA for pSB1C3	1000	1546	100	15.46	4.86
18	MVF-sicA for ColA	1000	1042	100	10.42	7.20

Not: 100ng pSB1C3 (2050 bp) ≈ 75 fmol

Digestion of „G-Blocks from IDT and pSB1C3“ (23.07.2015)

Digestion
	Insert	EcoRI-HF	PstI	NEB 2.1 Buffer	ddH₂O	Total
1x	10.0 ul	1.0 ul	1.0 ul	2.0 ul	6.0 ul	20.0 ul
17x	10.0 ul	17.0 ul	17.0 ul	34.0 ul	102.0 ul	20.0 ul

Digestion
	Vector	EcoRI-HF	PstI	NEB 2.1 Buffer	ddH₂O	Total
2x	17.0 ul	1.0 ul	1.0 ul	2.0 ul	-	21.0 ul

Result: Gel extraction was made.

GEL GÖRÜNTÜSÜ

PCR of “G-Blocks from IDT” (24.07.2015)

PCR from G-Bloks
	MgCl₂	(NH₄)2SO₄	CMV fwd	SV40 rev	tetR rev	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
3x (pTEToff)	7.5 ul	7.5 ul	1.5 ul	-	1.5 ul	1.5 ul	1.6 ul	36.9 ul		58.0 ul
15x	37.5 ul	37.5 ul	7.5 ul	7.5 ul	-	7.5 ul	3.0 ul	184.5 ul		285.0 ul

Cycling for 3x
	95˚C	95˚C	57˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Cycling for 15x
	95˚C	95˚C	60˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Results: Bands were at the expected section.

GEL GÖRÜNTÜSÜ

Gel Extraction (24.07.2015)

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	7/24/2015 12:40:09 PM	-0.8	ng/ul	-0.016	-0.020	0.79	0.24	DNA	50.00
2	pSB1C3	biospec	7/24/2015 12:41:56 PM	42.2	ng/ul	0.848	0.430	1.97	2.05	DNA	50.00

Ligation of „G-Blocks from IDT and pSB1C3“ (24.07.2015)

Ligation
	Insert DNA	Vector (pSB1C3)	T4 DNA Buffer	T4 DNA Ligase	ddH₂O	Total
1	4.9 ul	2.5 ul	2.0 ul	1.0 ul	9.8 ul	20.0 ul
2	7.2 ul	2.5 ul	2.0 ul	1.0 ul	7.3 ul	20.0 ul
3	9.2 ul	2.5 ul	2.0 ul	1.0 ul	5.3 ul	20.0 ul
4	4.8 ul	2.5 ul	2.0 ul	1.0 ul	9.7 ul	20.0 ul
5	4.8 ul	2.5 ul	2.0 ul	1.0 ul	9.7 ul	20.0 ul
6	8.4 ul	2.5 ul	2.0 ul	1.0 ul	6.1 ul	20.0 ul
7	5.8 ul	2.5 ul	2.0 ul	1.0 ul	8.7 ul	20.0 ul
8	4.9 ul	2.5 ul	2.0 ul	1.0 ul	9.6 ul	20.0 ul

RT 1h

Transformation was made.

(The results of psb1c3 gel extraction was lower. So we performed only the first 7 gene ligation.)

1	Toehold for cola
2	TnrA-pTnrA-RFP
3	ColA-KanR-dTer
4	HNS for PET
5	HNS-T108I for PET
6	potB59-pomA for PET
7	Gad E – for PET
8	TlpB for cola (NEB1)

Creating “Plasmid pCAG”

Digestion
	pTRE (1536 ng/ul)	pCAG (promoter) E	pCAG (promoter) Y	EcoRI	XhoI	Cut Smart Buffer	ddH₂O	Total
1	3.2 ul	-	-	0.5 ul	0.5 ul	2.0 ul	13.8 ul	20.0 ul	37˚C 2h
2	-	10 ul	-	0.5 ul	0.5 ul	2.0 ul	7.0 ul	20.0 ul	37˚C 2h
3	-	-	10 ul	0.5 ul	0.5 ul	2.0 ul	7.0 ul	20.0 ul	37˚C overnight

Bands were at the expected section. Gel extraction wil be made.

The Final Concentration

pCAG E: 21.5 ng/ul

pCAG Y: 12 ng/ul

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	eb	biospec	7/25/2015 5:03:57 AM	1.4	ng/ul	0.028	0.006	4.60	0.48	DNA	50.00
2	eb	biospec	7/25/2015 5:05:57 AM	0.0	ng/ul	0.000	-0.012	-0.03	0.06	DNA	50.00
3	ptre delta tre	biospec	7/25/2015 5:07:44 AM	194.0	ng/ul	3.880	2.033	1.91	2.20	DNA	50.00

Colony PCR of “pSB1C3 – GBlocks” (25.07.2015)

PCR from “pCAGGS”
	PCR MM	VR fwd	VR rev	ddH₂O	Tag	DNA	Total
1x	14.0 ul	1.0 ul	1.0 ul	7.5 ul	0.2 ul	5.0 ul	28.7 ul
33x	462.0 ul	33.0 ul	33.0 ul	247.5 ul	6.6 ul	165.0 ul	940.5 ul
33x	462.0 ul	33.0 ul	33.0 ul	247.5 ul	6.6 ul	165.0 ul	940.5 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Sonuc: 8-3 ve 4-9 bands were at the expected section. Liquid Culture was made.

GEL GÖRÜNTÜLERI

Colony PCR of “G-Blocks” (25.07.2015)

Colony PCR of “2,3,6,8”
	MgCl₂	(NH₄)2SO₄	CMV fwd	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	24.0 ul
6X	15.0 ul	15.0 ul	3.0 ul	3.0 ul	3.0 ul	1.2 ul	73.8 ul		114.0 ul

Colony PCR of “7,10,12,13”
	MgCl₂	(NH₄)2SO₄	CMV fwd	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	24.0 ul
6X	15.0 ul	15.0 ul	3.0 ul	3.0 ul	3.0 ul	1.2 ul	73.8 ul		114.0 ul

Colony PCR of “1,4,9,15”
	MgCl₂	(NH₄)2SO₄	CMV fwd	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	13.3 ul	5.0 ul	25.0 ul
6X	15.0 ul	15.0 ul	3.0 ul	3.0 ul	3.0 ul	1.2 ul	79.8 ul		120.0 ul

Colony PCR of “5,16”
	MgCl₂	(NH₄)2SO₄	CMV fwd	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	13.3 ul	5.0 ul	25.0 ul
2X	5.0 ul	5.0 ul	1.0 ul	1.0 ul	1.0 ul	0.4 ul	26.6 ul		40.0 ul

Cycling
	95˚C	95˚C	60˚C	72˚C	72˚C	Cycle
Time	5’	30’’	2’	1.5’	5’	35x

G-Blocks PCR / Gel Electrophoresis (25.07.2015)

PCR
	MgCl₂	(NH₄)2SO₄	CMV fwd	TetR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	13.3 ul	5.0 ul	25.0 ul
6X	15.0 ul	15.0 ul	3.0 ul	3.0 ul	3.0 ul	1.2 ul	79.8 ul		120.0 ul

Cycling
	95˚C	95˚C	60˚C	72˚C	72˚C	Cycle
Time	5’	30’’	2’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	7/26/2015 7:04:13 PM	0.8	ng/ul	0.016	-0.014	-1.13	1.36	DNA	50.00
2	blank	biospec	7/26/2015 7:05:51 PM	-0.4	ng/ul	-0.009	-0.017	0.52	0.17	DNA	50.00
3	colony pcr 8-3	biospec	7/26/2015 7:07:15 PM	87.9	ng/ul	1.758	0.908	1.94	2.09	DNA	50.00
4	colony pcr 4-9	biospec	7/26/2015 7:08:09 PM	101.4	ng/ul	2.027	1.089	1.86	1.74	DNA	50.00
5	colony pcr 4-9	biospec	7/26/2015 7:09:06 PM	71.4	ng/ul	1.429	0.749	1.91	1.96	DNA	50.00
6	colony pcr 8-3	biospec	7/26/2015 7:10:03 PM	87.9	ng/ul	1.758	0.910	1.93	2.10	DNA	50.00
7	colony pcr 4-9	biospec	7/26/2015 7:11:01 PM	57.1	ng/ul	1.142	0.596	1.91	1.77	DNA	50.00
8	colony pcr 4-9	biospec	7/26/2015 7:12:18 PM	77.2	ng/ul	1.543	0.820	1.88	1.76	DNA	50.00

Colony PCR of “pSB1C3- GBlocks” (26.07.2015)

Gradient PCR from pCAGGS
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	12.3 ul	5.0 ul	25.0 ul
41X	102.5 ul	102.5 ul	41.0 ul	41.0 ul	20.5 ul	504.3 ul		881.8 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

Digestion of “pTEToff” (26.07.2015)

Digestion
	pTEToff (1141 ng/ul)	SalI (Thermo)	HindIII (Thermo)	Fast Digest Buffer	ddH₂O	Total
Volume	3.1 ul	0.5 ul	0.5 ul	2.0 ul	13.9 ul	20.0 ul

37˚C 1h

Result: Bands were at the expected section. Gel purification was made.

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	7/26/2015 4:18:56 PM	0.3	ng/ul	0.005	-0.010	-0.56	-0.38	DNA	50.00
2	Ptetoff SalI hindIII	biospec	7/26/2015 4:20:23 PM	43.3	ng/ul	0.866	0.452	1.92	0.88	DNA	50.00

Creating “Plasmid pCAG” – Continue (27.07.2015)

Ligation of „pTRE TRE“ and „Digested pCAG“
	pTRE TRE (194 ng/ul)	pCAG E (21.5 ng/ul)	pCAG Y (12.0 ng/ul)	T4 DNA Ligase	Buffer	ddH₂O	Total
1	1.0 ul	2.0 ul	-	0.5 ul	2.0 ul	14.5 ul	20.0 ul
2	1.0 ul	-	3.6 ul	0.5 ul	2.0 ul	12.9 ul	20.0 ul

Result: On the first plate was six colonies. On the second plate was nine colonies.

Colony PCR will be made.

Colony PCR of „pCAG (plasmid)“

PCR
	MgCl₂	(NH₄)2SO₄	pTRE Luc fwd	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
16X	40.0 ul	40.0 ul	16.0 ul	16.0 ul	8.0 ul	3.2 ul	196.8 ul		320.0 ul

Cycling
	95˚C	95˚C	55˚C	72˚C	72˚C	Cycle
Time	5’	30’’	2’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

Colony PCR of “pSB1C3 – Gblocks” (27.07.2015)

PCR
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
57X	143.0 ul	143.0 ul	57.0 ul	57.0 ul	29.0 ul	11.4 ul	701.0 ul		1141.4 ul
57X	143.0 ul	143.0 ul	57.0 ul	57.0 ul	29.0 ul	11.4 ul	701.0 ul		1141.4 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Sonuc: Only 16-9 is positive.

GEL GÖRÜNTÜLERI

Colony PCR of “pSB1C3 – Gblocks” (28.07.2015)

Colony PCR of “2,3,8,9,11,13,14”
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
66X	165.0 ul	165.0 ul	66.0 ul	66.0 ul	33.0 ul	13.2 ul	811.8 ul		1650.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜLERI

Digestion of “HNS, HNS-T108I, potB59-pomA, GadE, TlpB, DAMP-Pex, Tev Protease” (27.07.2015)

Inserts from GBlocks were digested to ligate with PET45.

tab2wiki Tools Git Talk Powered by Wikimedia Labs Copy the following into the wiki - done!

	4	5	6	7	8	9	10
HNS (4)	10.0 ul	-	-	-	-	-	-
HNS-T108I (5)	-	10.0 ul	-	-	-	-	-
potB59-pomA (6)	-	-	10.0 ul	-	-	-	-
GadE (7)	-	-	-	10.0 ul	-	-	-
TlpB (8)	-	-	-	-	10.0 ul	-	-
DAMP-Pex (9)	-	-	-	-	-	10.0 ul	-
Tev Protease (10)	-	-	-	-	-	-	10.0 ul
Cut Smart Buffer	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul
₂XhoI	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul
BamHI-HF	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul
Milli-Q	7.0 ul	7.0 ul	7.0 ul	7.0 ul	7.0 ul	7.0 ul	7.0 ul

Total: 20 ul

37˚C overnight

Ligation will be made.

Ligation of “GBlocks (4,5,6,7,8,10)” and “PET45 (X+B)”

	4	5	6	7	8	10
Insert DNA	4.75 ul	4.75 ul	4.75 ul	4.75 ul	4.75 ul	4.75 ul
Vector (PET45)	2.20 ul	2.20 ul	.,20 ul	2.20 ul	2.20 ul	2.20 ul
Buffer	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul
T4 DNA Ligase	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul	0.5 ul
ddH₂O	10.55 ul	10.55 ul	10.55 ul	10.55 ul	10.55 ul	10.55 ul
Total	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul

Room Temperature 2h

	11	14
Insert DNA	3.4 ul	3.95 ul
Vector (PET45)	2.20 ul	2.20 ul
Buffer	2.0 ul	2.0 ul
T4 DNA Ligase	0.5 ul	0.5 ul
ddH₂O	11.9 ul	11.35 ul
Total	20.0 ul	20.0 ul

Room Temperature 2h

Creating of “pTRE – mLacI miRNA-BS” (27.07.2015)

Digestion of “pTRE” and G-Blocks”
	pTRE	EcoRI-HF	BamHI-HF	mLacI-miR 373 BS	mLacI-miR (21-223) BS	Cut Smart	ddH₂O	Total
1	3.1 ul	0.5 ul	0.5 ul	-	-	2.0 ul	13.9 ul	20.0 ul	37˚C-3h Gel Elect.
2	-	0.5 ul	0.5 ul	10.0 ul	-	2.0 ul	7.0 ul	20.0 ul	37˚C overnight/80˚C-10’ heat inactivation
3	-	0.5 ul	0.5 ul	-	10.0 ul	2.0 ul	7.0 ul	20.0 ul	37˚C overnight/80˚C-10’ heat inactivation

Gel purification was made.

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	7/27/2015 2:26:05 PM	0.3	ng/ul	0.007	-0.004	-1.71	0.20	DNA	50.00
2	pTRE e+b	biospec	7/27/2015 2:27:08 PM	92.9	ng/ul	1.859	1.015	1.83	1.24	DNA	50.00

Creating of “pTRE – mLacI miRBS” (27.07.2015)

Ligation of „pTRE” and “mLacI miRBS”
	Digested pTRE	D. mLacI miR373 BS	D. mLacI miR(21-223) BS	T4 DNA Ligase	T4 DNA Buffer	ddH₂O	Total
1	1.0 ul	5.9 ul	-	2.0 ul	0.5 ul	10.6 ul	20.0 ul
2	1.0 ul	-	5.9 ul	2.0 ul	0.5 ul	10.6 ul	20.0 ul

Room Temperature 2h

Transformation was made with TOP10.

Creating of “pTEToff – miRBS” (27.07.2015)

Digestion
	pTEToff (2.5 ng/ul)	miRNA switch miR373 BS	miRNA switch miR(223-21) BS	SalI (FD)	HindIII (FD)	Fast Digest Buffer	ddH₂O	Total
1	2.0 ul	-	-	0.5 ul	0.5 ul	5.0 ul	42.0 ul	50.0 ul	37˚C-3h Gel Elect.
2	-	10.0 ul	-	0.5 ul	0.5 ul	2.0 ul	7.0 ul	20.0 ul	37˚C overnight/80˚C-10’ heat inactivation
3	-	-	10.0 ul	0.5 ul	0.5 ul	2.0 ul	7.0 ul	20.0 ul	37˚C overnight/80˚C-10’ heat inactivation

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	7/27/2015 8:35:05 PM	0.0	ng/ul	0.000	-0.013	-0.01	0.01	DNA	50.00
2	pTRE s+h	biospec	7/27/2015 8:36:16 PM	76.7	ng/ul	1.534	0.810	1.89	1.05	DNA	50.00

Creating of “pTEToff – miRNA Switch miR BS” (28.07.2015)

Ligation of „pTRE” and “mLacI miRBS”
	Digested pTEToff	D. mLacI miR373 BS	D. mLacI miR(21-223) BS	T4 DNA Ligase	T4 DNA Buffer	ddH₂O	Total
1	2.2 ul	3.4 ul	-	2.0 ul	0.5 ul	10.9 ul	20.0 ul
2	2.2 ul	-	4.0 ul	2.0 ul	0.5 ul	11.3 ul	20.0 ul

Room Tempretare 2h

Transformation was made.

Creating of “pCAG”

Digestion of “pCAG (Promotor)”
	pCAG (promoter) E	pCAG (promoter) Y	EcoRI-HF	XhoI-HF	Cut Smart Buffer	ddH₂O	Total
1	10.0 ul	-	0.5 ul	0.5 ul	2.0 ul	7.0 ul	20.0 ul	37˚C overnight
2	-	10.0 ul	0.5 ul	0.5 ul	2.0 ul	7.0 ul	20.0 ul	37˚C overnight

Mini Prep of “Colony PCR 16-9”

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	7/28/2015 12:11:17 PM	-1.9	ng/ul	-0.038	-0.049	0.77	0.19	DNA	50.00
2	blank	biospec	7/28/2015 12:12:22 PM	-0.4	ng/ul	-0.007	-0.014	0.53	0.30	DNA	50.00
3	colony pcr 16-9	biospec	7/28/2015 12:13:33 PM	84.9	ng/ul	1.698	0.881	1.93	2.02	DNA	50.00
4	colony pcr 16-9	biospec	7/28/2015 12:14:24 PM	76.3	ng/ul	1.527	0.797	1.92	1.95	DNA	50.00

Mini Prep of “pTEToff”

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	7/30/2015 4:44:01 PM	0.4	ng/ul	0.008	-0.005	-1.55	1.11	DNA	50.00
2	ptetoff neb	biospec	7/30/2015 4:45:00 PM	442.2	ng/ul	8.884	4.683	1.89	2.20	DNA	50.00
3	ptetoff bl21	biospec	7/30/2015 4:46:08 PM	314.3	ng/ul	6.286	3.317	1.90	2.22	DNA	50.00

Cut Check of “HNS toehold and Trigger RNA” (30.07.2015)

	HNS (70 ng/ul)	TlpB (87 ng/ul)	Trigger RNA (80 ng/ul)	EcoRI (Thermo)	PstI (Thermo)	Fast Digest Buffer	ddH₂O	Total
1	3.5 ul	-	-	1.0 ul	1.0 ul	2.0 ul	12.5 ul	20.0 ul	37˚C 20min
2	-	3.0 ul	-	1.0 ul	1.0 ul	2.0 ul	13.0 ul	20.0 ul	37˚C 20min
3	-	-	3.0 ul	1.0 ul	1.0 ul	2.0 ul	13.0 ul	20.0 ul	37˚C 20min

Gel Electropheres was made.

Result: Bands were at the expected section.

HNS: 480 bp

tgRNA:˞ 100bp

Toehold: ˃1000bp

GEL GÖRÜNTÜSÜ

Colony PCR of „9-10 GBlocks“ (31.07.2015)

Result: negative

Expected: ˞1100 bp

Observed: ˞300 bp

Gel extraction 1-6

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/2/2015 12:01:41 AM	0.2	ng/ul	0.004	-0.002	-2.02	17.14	DNA	50.00
2	psb1c3	biospec	8/2/2015 12:31:14 AM	43.0	ng/ul	0.861	0.462	1.87	0.94	DNA	50.00

Gel extraction 8-9

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/2/2015 12:27:36 AM	-0.4	ng/ul	-0.009	-0.012	0.74	-0.09	DNA	50.00
2	psb1c3 atoms	biospec	8/2/2015 12:34:11 AM	10.4	ng/ul	0.208	0.111	1.87	0.03	DNA	50.00

Colony PCR of „pSB1C3 – Gblocks“

Colony PCR
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	13.3 ul	5.0 ul	25.0 ul
17X	42.5 ul	42.5 ul	8.5 ul	8.5 ul	8.5 ul	3.4 ul	226.1 ul		340.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜLERI

August

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

Week 1, 01.08.-02.08.

Mini Prep of “pSB1C3-RFP”

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/1/2015 6:03:37 PM	-0.1	ng/ul	-0.003	-0.009	0.30	0.12	DNA	50.00
2	psb1c3-A	biospec	8/1/2015 6:06:00 PM	207.0	ng/ul	4.140	2.184	1.90	2.11	DNA	50.00
3	psb1c3-B	biospec	8/1/2015 6:07:07 PM	209.0	ng/ul	4.179	2.210	1.89	2.19	DNA	50.00
4	psb1c3-C	biospec	8/1/2015 6:08:05 PM	157.3	ng/ul	3.147	1.664	1.89	2.09	DNA	50.00
5	psb1c3-D	biospec	8/1/2015 6:08:58 PM	242.5	ng/ul	4.851	2.548	1.90	2.11	DNA	50.00
6	psb1c3-E	biospec	8/1/2015 6:09:42 PM	116.4	ng/ul	2.329	1.215	1.92	2.17	DNA	50.00
7	psb1c3-F	biospec	8/1/2015 6:10:28 PM	236.4	ng/ul	4.729	2.503	1.89	2.17	DNA	50.00
8	psb1c3-71	biospec	8/1/2015 6:11:33 PM	111.7	ng/ul	2.233	1.175	1.90	2.10	DNA	50.00
9	psb1c3-72	biospec	8/1/2015 6:12:14 PM	106.4	ng/ul	2.127	1.100	1.93	2.23	DNA	50.00
10	psb1c3-8	biospec	8/1/2015 6:12:51 PM	90.7	ng/ul	1.814	0.959	1.89	1.98	DNA	50.00

Ligation of „pSB1C3 – Gblocks“ (02.08.2015)

	2	3	5	6	7	8	9	10	11	12	13	14	15
Insert	7.3 ul	7.0 ul	4.8 ul	8.4 ul	5.8 ul	8.3 ul	3.6 ul	3.9 ul	3.4 ul	4.4 ul	5.9 ul	3.9 ul	5.9 ul
Vector	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul
Buffer	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul	2.0 ul
Enzyme	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul	1.0 ul
ddH₂O	7.7 ul	8.0 ul	10.2 ul	6.6 ul	9.2 ul	6.7 ul	11.4 ul	11.1 ul	11.6 ul	10.6 ul	9.1 ul	11.1 ul	9.1 ul
Total	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul	20.0 ul

Week 2, 03.08.-09.08.

Colony PCR of „pSB1C3 – Gblocks“ (03.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
22X	55.0 ul	55.0 ul	22.0 ul	22.0 ul	11.0 ul	4.4 ul	271.0 ul		440.4 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Sonuc: negative

GEL GÖRÜNTÜSÜ

Colony PCR of „pSB1C3 – Gblocks #14“ (03.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	VR fwd	VR rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
36X	90.0 ul	90.0 ul	36.0 ul	36.0 ul	18.0 ul	7.2 ul	442.8 ul		720.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Sonuc: negative

GEL GÖRÜNTÜSÜ

Digestion of “pSB1C3-RFP” (03.08.2015)

Digestion of “pSB1C3-RFP”
	Vector	Fast Digest Buffer	EcoRI (FD)	PstI (FD)	ddH₂O	Total
1x	2.0 ul	2.0 ul	1.0 ul	1.0 ul	14.0 ul	20.0 ul

37˚C 2h

Gel Electropheresis was made.

Result: Bands were at the expected section. Gel purification was made.

GEL GÖRÜNTÜSÜ

Ligation of “pSB1C3 – Gblock #14”

Ligation of “pSB1C3 – Gblock #14”
	Insert	Vector	T4 DNA Ligase Buffer	T4 DNA Ligase	ddH₂O	Total
1) PCR clean up vektörü ile 2.5 ul	2.5 ul	2.0 ul
(100ng)	2.0 ul
	1.0 ul	12.5 ul	20.0 ul
2) Gel Extraction 7.5 ul	7.5 ul	0.8 ul
(50ng)	2.0 ul
	1.0 ul	8.7 ul	20.0 ul

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/3/2015 6:10:02 PM	0.2	ng/ul	0.004	-0.010	-0.41	1.13	DNA	50.00
2	psb1c3 gel	biospec	8/3/2015 6:12:16 PM	62.5	ng/ul	1.250	0.664	1.88	0.90	DNA	50.00
4	psb1c3 clean-up	biospec	8/3/2015 6:13:39 PM	170.5	ng/ul	3.409	1.884	1.81	1.33	DNA	50.00

PCR of “G-Bloks via Q5 Polymerase” (04.08.2015)

PCR
	SV40 poly A rev	CMV fwd	tetR rev	2x Q5 Master Mix	DNA	Total
1x	2.5 ul	2.5 ul	2.5 ul	10.0 ul	5.0 ul	20.0 ul

Cycling SV40 rev/CMV fwd
	95˚C	95˚C	63˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1’	2’	35x

Cycling CMV fwd/tetR rev
	98˚C	98˚C	66˚C	72˚C	72˚C	Cycle
Time	30’’	10’’	30’’	1’	2’	35x

Digestion of “pSB1C3-RFP” (04.08.2015)

Digestion
	Vector	Cut Smart Buffer	EcoRI	SpeI	ddH₂O	Total
1x	2.0 ul	2.0 ul	1.0 ul	1.0 ul	14.0 ul	20.0 ul

37˚C 4h

Result: Bands were at the expected section.

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/4/2015 7:03:09 AM	0.9	ng/ul	0.017	0.005	3.41	0.68	DNA	50.00
2	Blank2	biospec	8/4/2015 7:04:51 AM	-0.5	ng/ul	-0.010	-0.017	0.59	0.45	DNA	50.00
4	clean up	biospec	8/4/2015 7:05:55 AM	86.8	ng/ul	1.735	0.913	1.90	0.40	DNA	50.00
5	gel ext	biospec	8/4/2015 7:06:51 AM	36.2	ng/ul	0.723	0.380	1.90	0.29	DNA	50.00

Colony PCR of “11,12,13 pSB1C3 - GBlocks” (04.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	VF	VR	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.2 ul	13.3 ul	5.0 ul	25.0 ul
17X	42.5 ul	42.5 ul	8.5 ul	8.5 ul	3.4 ul	226.1 ul		340.0 ul

Cycling CMV fwd/tetR rev
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

Ligation and Transformation of “5,6,7,8,9 pSB1C3-GBlocks” (04.08.2015)

Ligation of “GBlocks 11-12 pSB1C3”
	Vector	Gen	Buffer	Ligase	ddH₂O	Total
5	0.8 ul	2.37 ul	1.0 ul	0.5 ul	5.33 ul	10.0 ul
6	0.8 ul	4.2 ul	1.0 ul	0.5 ul	3.5 ul	10.0 ul
7	0.8 ul	2.94 ul	1.0 ul	0.5 ul	4.76 ul	10.0 ul
8	0.8 ul	4.16 ul	1.0 ul	0.5 ul	3.54 ul	10.0 ul
9	0.8 ul	1.79 ul	1.0 ul	0.5 ul	5.91 ul	10.0 ul

50ng DNA

1 vector:0.5 insert

Transfomation with TOP10.

Ligation of “GBlocks 11-12 pSB1C3” (05.08.2015)

Ligation of “GBlocks 11-12 pSB1C3”
	pSB1C3	Insert	Buffer	Ligase	ddH₂O	Total
11	0.8 ul	1.7 ul	1.0 ul	0.5 ul	6.0 ul	10.0 ul
12	0.4 ul	1.1 ul	1.0 ul	0.5 ul	7.0 ul	10.0 ul

Transformation was made.

Colony PCR of “5,6,7,8,9,T7 system – Gblocks” (05.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	VR	VF	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.1 ul	13.3 ul	5.0 ul	24.9 ul
58X	145.0 ul	145.0 ul	29.0 ul	29.0 ul	29.0 ul	5.8 ul	771.4 ul		1154.2 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: ?????????????

GEL GÖRÜNTÜSÜ

Ligation of “GBlocks 2,5,6,7,8,9,10,12,13,15,17 pSB1C3-RFP” (06.08.2015)

Ligation of “GBlocks 11-12 pSB1C3”
	pSB1C3	Insert	Buffer	Ligase	ddH₂O	Total
2	1.4 ul	3.65 ul	1.0 ul	0.5 ul	3.45 ul	10.0 ul
5	1.4 ul	2.4 ul	1.0 ul	0.5 ul	4.7 ul	10.0 ul
6	1.4 ul	4.2 ul	1.0 ul	0.5 ul	2.9 ul	10.0 ul
7	1.4 ul	2.9 ul	1.0 ul	0.5 ul	4.2 ul	10.0 ul
8	1.4 ul	4.15 ul	1.0 ul	0.5 ul	2.95 ul	10.0 ul
9	1.4 ul	1.8 ul	1.0 ul	0.5 ul	5.3 ul	10.0 ul
10	1.4 ul	1.9 ul	1.0 ul	0.5 ul	5.2 ul	10.0 ul
12	1.4 ul	2.2 ul	1.0 ul	0.5 ul	4.9 ul	10.0 ul
13	1.4 ul	2.9 ul	1.0 ul	0.5 ul	4.2 ul	10.0 ul
15	1.4 ul	2.95 ul	1.0 ul	0.5 ul	4.15 ul	10.0 ul
17	1.4 ul	1.25 ul	1.0 ul	0.5 ul	5.75 ul	10.0 ul

Transformation was made.

Colony PCR of “pTRE-1,13,15” (06.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	CMV fwd	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
20X	50.0 ul	50.0 ul	20.0 ul	20.0 ul	10.0 ul	2.0 ul	246.0 ul		400.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

Colony PCR of “5,6,7,9 – Gblocks” (06.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	VR	VF	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	0.5 ul	0.5 ul	0.5 ul	0.2 ul	13.3 ul	5.0 ul	25.0 ul
40X	100.0 ul	100.0 ul	20.0 ul	20.0 ul	20.0 ul	8.0 ul	532.0 ul		800.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

Colony PCR of “7,8,10 – Gblocks” from Masterplate (08.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	T7 terminatör Rev.	T7 terminatör For.	dNTP	Tag	ddH₂O	DNA	Totalzz
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.5 ul	12.3 ul	5.0 ul	25.0 ul
26X	65.0 ul	65.0 ul	26.0 ul	26.0 ul	13.0 ul	13.0 ul	319.8 ul		527.8 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ????

Digestion of “pET45” (09.08.2015)

Digestion
	pET45 (455ng/ul)	pSB1C3-RFP (400 ng/ul)	Cut Smart Buffer	XhoI (Neb)	BamHI-HF (Neb)	EcoRI-HF (Neb)	SpeI (Neb)	ddH₂O	Total
1	-	5.0 ul	2.0 ul	-	-	1.0 ul	1.0 ul	11.0 ul	20.0 ul
2	4.0 ul	-	2.0 ul	1.0 ul	1.0 ul	-	-	12.0 ul	20.0 ul

37˚C 3.5h

Result: Positive. Gel extraction was made.

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/9/2015 5:04:14 PM	-0.6	ng/ul	-0.011	0.005	3.41	0.68	DNA	50.00
2	pet45 x+b	biospec	8/9/2015 5:05:26 PM	106.9	ng/ul	2.138	-0.017	0.59	0.45	DNA	50.00
4	psb1c3 e+s	biospec	8/9/2015 5:06:20 PM	133.3	ng/ul	2.666	0.913	1.90	0.40	DNA	50.00

Week 3, 10.16.-10.16.

Ligation of “PET45 – HNS” (10.08.2015)

Ligation
	Vector	Insert	T4 DNA Ligase Buffer	T4 DNA Ligase	ddH₂O	Total
1x	2.1 ul	1.0 ul	2.0 ul	0.5 ul	14.4 ul	20.0 ul

16˚C 2h

Digestion of “pSB1C3 – HNS”

Ligation
	Gen	BamHI	XhoI	Cut Smart Buffer	ddH₂O	Total
1x	11.5 ul	1.0 ul	1.0 ul	2.0 ul	4.5 ul	20.0 ul

37˚C 3h

Colony PCR of “pTRE #13”

Colony PCR
	MgCl₂	(NH₄)2SO₄	CMV for.	SV40 rev.	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
10X	25.0 ul	25.0 ul	10.0 ul	10.0 ul	5.0 ul	2.0 ul	123.0 ul		250.0 ul

Cycling
	95˚C	95˚C	60˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

Colony PCR of “PET45-4,5,6,7,8,9,10” (10.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	T7 ter. fw	T7 ter. rev	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
86X	215.0 ul	215.0 ul	86.0 ul	186.0 ul	43.0 ul	17.2 ul	1075.0 ul		1720.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: Bands were at the expected section. Liquid Culture was made.

GEL GÖRÜNTÜLERI

Digestion of „pColA-KanR and „pSB1C3-Toehold“ (10.08.2015)

Digestion
	pColA-KanR (500 ng/ul)	pSB1C3-Toehold (500 ng/ul)	NotI-HF (Neb)	Cut Smart Buffer	ddH₂O	Total
1	11.6 ul	-	1.0 ul	2.0 ul	5.4 ul	20.0 ul
2	-	6.5 ul	1.0 ul	2.0 ul	10.5 ul	20.0 ul

37˚C 2h

pColA-KanR: 1.0 ul rSAP/37˚C 1h/-80˚C 20’’ heat shock

Sonuc:?????

GEL GÖRÜNTÜSÜ

Ligation of “pColA-KanR and pSB1C3-Toehold”

Ligation
	pColA-KanR (500 ng/ul)	pSb1C3-Toehold	T4 DNA Ligase Buffer (Thermo)	T4 DNA Ligase (Thermo)	ddH₂O	Total	Vector:Insert Ratios
1	2.0 ul	1.0 ul	2.0 ul	1.0 ul	14.0 ul	20.0 ul	1:0.33
2	2.0 ul	3.0 ul	2.0 ul	1.0 ul	12.0 ul	20.0 ul	1:1

22˚C 1h

Mini Prep of “pColA-3,4,5”

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/11/2015 11:11:42 PM	-0.2	ng/ul	-0.003	-0.007	0.49	0.13	DNA	50.00
2	ColA-3	biospec	8/11/2015 11:13:07 PM	27.2	ng/ul	0.544	0.260	2.10	1.95	DNA	50.00
3	ColA-4	biospec	8/11/2015 11:14:38 PM	25.5	ng/ul	0.509	0.253	2.01	1.82	DNA	50.00
4	ColA-4(2)	biospec	8/11/2015 11:15:58 PM	25.8	ng/ul	0.517	0.255	2.022	1.81	DNA	50.00
5	ColA-5	biospec	8/11/2015 11:17:02 PM	44.4	ng/ul	0.887	0.449	1.98	1.76	DNA	50.00

Result: The Band of pColA were at the expected section. 2000 pb

(CUT CHECK GÖRÜNTÜSÜ)(12.08.2015)

Ligation of “#2 GBlock with pET45” (11.08.2015)

Ligation
	pET45 (100 ng/ul)	#2 GBlock	T4 DNA Ligase Buffer	T4 DNA Ligase	ddH₂O	Total
1x	1.0 ul	7.3 ul	2.0 ul	1.0 ul	8.7 ul	20.0 ul

22˚C - 1’40’’

Transformation was made.

Mini Prep

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/12/2015 7:38:28 PM	0.4	ng/ul	0.008	-0.001	-8.91	0.76	DNA	50.00
2	pet45 4-4	biospec	8/12/2015 7:40:21 PM	40.3	ng/ul	0.807	0.402	2.01	2.44	DNA	50.00
3	pet45 4-3	biospec	8/12/2015 7:41:10 PM	53.1	ng/ul	1.063	0.570	1.86	1.74	DNA	50.00
4	pet45 8-12	biospec	8/12/2015 7:41:55 PM	61.8	ng/ul	1.236	0.642	1.92	2.08	DNA	50.00
5	pet45 8-3	biospec	8/12/2015 7:42:37 PM	32.9	ng/ul	0.658	0.332	1.98	2.00	DNA	50.00
6	pet45 9-11	biospec	8/12/2015 7:44:03 PM	34.5	ng/ul	0.690	0.345	2.00	1.77	DNA	50.00
7	pet45 8-1	biospec	8/12/2015 7:44:54 PM	39.0	ng/ul	0.780	0.408	1.91	1.72	DNA	50.00
8	pet45 5-10	biospec	8/12/2015 7:45:49 PM	43.2	ng/ul	0.865	0.441	1.96	1.81	DNA	50.00
9	pet45 6-3	biospec	8/12/2015 7:46:42 PM	43.5	ng/ul	0.870	0.437	1.99	1.92	DNA	50.00
10	pet45 9-8	biospec	8/12/2015 7:47:23 PM	48.1	ng/ul	0.961	0.496	1.94	1.88	DNA	50.00
11	pet45 7-4	biospec	8/12/2015 7:48:06 PM	36.2	ng/ul	0.723	0.372	1.94	1.97	DNA	50.00
12	pet45 4-1	biospec	8/12/2015 7:48:50 PM	49.4	ng/ul	0.987	0.509	1.94	1.99	DNA	50.00
13	pet45 4-2	biospec	8/12/2015 7:49:43 PM	59.3	ng/ul	1.187	0.606	1.96	2.09	DNA	50.00
14	pet45 7-3	biospec	8/12/2015 7:50:33 PM	30.2	ng/ul	0.604	0.307	1.97	1.96	DNA	50.00
15	psb1c3 12	biospec	8/12/2015 7:51:18 PM	141.1	ng/ul	2.823	1.462	1.93	2.14	DNA	50.00
16	pet45 5-7	biospec	8/12/2015 7:52:04 PM	26.1	ng/ul	0.522	0.265	1.97	2.00	DNA	50.00
17	pet45 5-3	biospec	8/12/2015 7:52:49 PM	46.1	ng/ul	0.923	0.470	1.96	2.02	DNA	50.00
18	pet45 6-12	biospec	8/12/2015 7:53:36 PM	29.3	ng/ul	0.586	0.283	2.07	1.80	DNA	50.00
19	pet45 1-12	biospec	8/12/2015 7:54:25 PM	21.4	ng/ul	0.428	0.210	2.04	1.72	DNA	50.00
20	pet45 5-8	biospec	8/12/2015 7:55:08 PM	29.1	ng/ul	0.582	0.299	1.95	2.07	DNA	50.00
21	psb1c3 1	biospec	8/12/2015 7:55:51 PM	75.3	ng/ul	1.507	0.779	1.93	2.07	DNA	50.00
22	pet45 8-4	biospec	8/12/2015 7:56:33 PM	44.1	ng/ul	0.882	0.451	1.96	1.99	DNA	50.00
23	pet45 7-2	biospec	8/12/2015 7:57:16 PM	25.3	ng/ul	0.506	0.259	1.95	1.70	DNA	50.00

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/12/2015 7:17:01 PM	-0.3	ng/ul	-0.007	-0.015	0.47	0.23	DNA	50.00
2	psb1c3-4hns	biospec	8/12/2015 7:18:16 PM	61.9	ng/ul	1.237	0.652	1.90	2.17	DNA	50.00
3	pet45 10-1	biospec	8/12/2015 7:19:32 PM	46.7	ng/ul	0.934	0.486	1.92	2.00	DNA	50.00
4	pet45 10-6	biospec	8/12/2015 7:20:23 PM	52.0	ng/ul	1.040	0.549	1.89	1.88	DNA	50.00
5	pet45 10-3	biospec	8/12/2015 7:21:14 PM	51.9	ng/ul	1.037	0.536	1.94	2.10	DNA	50.00
6	pet45 9-6	biospec	8/12/2015 7:22:10 PM	37.7	ng/ul	0.755	0.389	1.94	1.99	DNA	50.00
7	pet45 10-8	biospec	8/12/2015 7:23:08 PM	85.3	ng/ul	1.705	1.002	1.70	0.93	DNA	50.00
8	psb1c3 16-trigger rna	biospec	8/12/2015 7:24:27 PM	125.7	ng/ul	2.514	1.401	1.79	1.32	DNA	50.00
9	pet45 10	biospec	8/12/2015 7:25:23 PM	84.7	ng/ul	1.694	1.025	1.65	0.76	DNA	50.00
10	pet45 6-6	biospec	8/12/2015 7:26:23 PM	100.3	ng/ul	2.006	1.124	1.79	1.24	DNA	50.00
11	pet45 8-2	biospec	8/12/2015 7:27:20 PM	67.5	ng/ul	1.349	0.778	1.73	0.91	DNA	50.00
12	pet45 7-1	biospec	8/12/2015 7:28:07 PM	52.5	ng/ul	1.050	0.549	1.91	1.83	DNA	50.00

Cut Check of “pET45 1,4,5,6,7,8,9,10 – BamHI/XhoI” (13.08.2015)

Ligation
	Gen (250 ng/ul)	Cut Smart Buffer	BamHI (NEB)	XhoI (NEB)	ddH₂O	Total
1	11.7 ul	2.0 ul	1.0 ul	1.0 ul	4.3 ul	20.0 ul
4	6.2 ul	2.0 ul	1.0 ul	1.0 ul	9.8 ul	20.0 ul
5	8.6 ul	2.0 ul	1.0 ul	1.0 ul	7.4 ul	20.0 ul
6	8.6 ul	2.0 ul	1.0 ul	1.0 ul	7.4 ul	20.0 ul
7	8.3 ul	2.0 ul	1.0 ul	1.0 ul	7.7 ul	20.0 ul
8	7.4 ul	2.0 ul	1.0 ul	1.0 ul	8.6 ul	20.0 ul
9	6.7 ul	2.0 ul	1.0 ul	1.0 ul	9.3 ul	20.0 ul
10	5.9 ul	2.0 ul	1.0 ul	1.0 ul	10.1 ul	20.0 ul

Dogru cikan koloniler BL21 bakterisine transformasyon edildi ve sivi kültür yapildi. Sonrasinda protein izolasyon ve WB yapilacak.

GEL GÖRÜNTÜLERI

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/13/2015 1:31:59 AM	0.0	ng/ul	-0.001	-0.005	0.17	0.08	DNA	50.00
2	psb1c3-e+p	biospec	8/13/2015 1:32:58 AM	58.6	ng/ul	1.172	0.618	1.90	0.57	DNA	50.00

Colony PCR of “pTRE – 12,13 GBlocks” (14.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	CMV fw	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
15X	37.5 ul	37.5 ul	15.0 ul	15.0 ul	7.5 ul	3.0 ul	184.5 ul		300.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Sonuc: negative

GEL GÖRÜNTÜSÜ

Colony PCR of “pColA – Toehold GFP” (14.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	pColA fw	pColA rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
26X	65.0 ul	65.0 ul	26.0 ul	26.0 ul	13.0 ul	5.2 ul	319.8 ul		520.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: 2-4, 2-8, 1-6 Bands were at the expected section.

Digestion of “pSB1C3-RFP” (14.08.2015)

Digestion
	pSB1C3-RFP (400 ng/ul)	Cut Smart Buffer	EcoRI-HF	PstI	ddH₂O	Total
1	5.0 ul	2.0 ul	1.0 ul	1.0 ul	11.0 ul	20.0 ul
2	5.0 ul	2.0 ul	1.0 ul	1.0 ul	11.0 ul	20.0 ul
3	5.0 ul	2.0 ul	1.0 ul	1.0 ul	11.0 ul	20.0 ul

37˚C – 3.5h

Gel extraction was made.

Ligation of “pSB1C3-GBlocks”

Ligation
	Digested pSB1C3	GBlocks	T4 DNA Ligase Buffer	T4 DNA Ligase	ddH₂O	Total
1	1.7 ul	???????	2 n.0 ul	1.0 ul	???????	20.0 ul

22˚C – 1.30h

Transformation with Neb10-β. Colony PCR will be made.

Colony PCR of “GBlocks – 2,5,6,7,8,9” (14.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	VR	VF	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
45X	112.5 ul	112.5 ul	45.0 ul	45.0 ul	22.5 ul	9.0 ul	553.5 ul		900.0 ul
55X	137.5 ul	137.5 ul	55.0 ul	55.0 ul	27.5 ul	11.0 ul	676.5 ul		1100.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Sonuc:??????

GEL GÖRÜNTÜLERI

Digestion/Gel Electrophoresis/Gel Extraction and Purification of “pSB1C3” (16.08.2015)

Digestion
	DNA	FD	EcoRI	PstI	ddH₂O	Total
1x	2.5 ul	2.0 ul	1.0 ul	1.0 ul	13.5 ul	20.0 ul

37˚C 1h

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/16/2015 10:45:30 PM	0.1	ng/ul	0.003	-0.017	0.17	-0.14	DNA	50.00
2	psb1c3-e+p	biospec	8/16/2015 10:47:18 PM	14.0	ng/ul	0.279	0.145	1.92	0.15	DNA	50.00

Colony PCR of “pSB1C3 7,10,12,13 and pET45 2” (14.08.2015)

Colony PCR of “pSB1C3 7,10,12,13”
	MgCl₂	(NH₄)2SO₄	VR	VF	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
6X	15.0 ul	15.0 ul	6.0 ul	6.0 ul	3.0 ul	1.2 ul	73.8 ul		120.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

Colony PCR of “pET45 2”
	MgCl₂	(NH₄)2SO₄	VR	VF	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
12X	30.0 ul	30.0 ul	12.0 ul	12.0 ul	6.0 ul	2.4 ul	147.6 ul		240.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/16/2015 8:54:05 PM	-0.2	ng/ul	-0.004	-0.017	0.25	0.12	DNA	50.00
2	psb1c3 13-15	biospec	8/16/2015 8:55:29 PM	125.2	ng/ul	2.505	1.339	1.87	2.01	DNA	50.00
3	psb1c3 15-19	biospec	8/16/2015 8:56:24 PM	93.5	ng/ul	1.871	0.999	1.87	1.94	DNA	50.00
4	psb1c3 7-16	biospec	8/16/2015 8:57:33 PM	149.4	ng/ul	2.988	1.575	1.90	2.14	DNA	50.00
5	psb1c3 10-16	biospec	8/16/2015 8:58:18 PM	138.0	ng/ul	2.760	1.469	1.88	2.13	DNA	50.00
6	psb1c3 10-17	biospec	8/16/2015 8:59:03 PM	185.5	ng/ul	3.710	1.994	1.86	1.86	DNA	50.00
7	psb1c3 7-11	biospec	8/16/2015 8:00:54 PM	73.8	ng/ul	1.475	0.843	1.75	1.08	DNA	50.00
8	psb1c3 13-14	biospec	8/16/2015 8:01:53 PM	178.7	ng/ul	3.575	1.922	1.86	1.87	DNA	50.00
9	psb1c3 7-20	biospec	8/16/2015 8:02:54 PM	73.6	ng/ul	1.472	0.774	1.90	2.05	DNA	50.00
10	pcola toe 2-4	biospec	8/16/2015 8:03:57 PM	40.1	ng/ul	0.801	0.441	1.82	1.13	DNA	50.00
11	pcola toe 2-8	biospec	8/16/2015 8:05:31 PM	48.7	ng/ul	0.974	0.503	1.94	1.61	DNA	50.00
12	pcola toe 1-6	biospec	8/16/2015 8:06:09 PM	54.9	ng/ul	1.098	0.570	1.93	1.62	DNA	50.00
13	pcola blunt 1	biospec	8/16/2015 8:06:52 PM	23.9	ng/ul	0.479	0.244	1.96	1.24	DNA	50.00
14	pcola blunt 6	biospec	8/16/2015 8:07:30 PM	31.1	ng/ul	0.623	0.314	1.98	1.67	DNA	50.00

Week 4, 17.08.-23.08.

Ligation of “pColA and pSB1C3 #9 (Damp-Pex)” (18.08.2015)

Ligation
	pColA (Vector 50 ng/ul)	Insert (Damp-Pex)	T4 DNA Ligase Buffer	T4 DNA Ligase	ddH₂O	Total	Vector:Insert Ratio
9.1	2.0 ul	1.0 ul	2.0 ul	1.0 ul	14.0 ul	20.0 ul	1:0.33
9.2	2.0 ul	2.8 ul	2.0 ul	1.0 ul	12.2 ul	20.0 ul	1:1

Miniprep of “pTet-off DH5α /Top10/NEB5fα/NEB10β”

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/18/2015 3:04:27 PM	0.4	ng/ul	0.008	-0.008	-1.01	1.01	DNA	50.00
2	top10	biospec	8/18/2015 3:05:19 PM	258.5	ng/ul	5.169	2.753	1.88	2.11	DNA	50.00
3	neb5a	biospec	8/18/2015 3:06:25 PM	30.6	ng/ul	0.612	0.324	1.89	1.34	DNA	50.00
4	neb5a	biospec	8/18/2015 3:07:30 PM	33.4	ng/ul	0.668	0.350	1.91	1.27	DNA	50.00
5	neb10beta	biospec	8/18/2015 3:08:22 PM	214.6	ng/ul	4.292	2.264	1.90	2.19	DNA	50.00

Colony PCR of “pET45 #2.8” (15.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	VR	VF	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
25X	62.5 ul	62.5 ul	25.0 ul	25.0 ul	12.5 ul	5.0 ul	307.5 ul		500.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

Colony PCR of “pSB1C3 17,6,8” (S. 118/15.08.2015)

Colony PCR of “pSB1C3 17,6,8”
	MgCl₂	(NH₄)2SO₄	VR	VF	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
32X	80.0 ul	80.0 ul	32.0 ul	32.0 ul	16.0 ul	6.4 ul	393.6 ul		640.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: 6: Liquid Culture was made.

8: sequencing will be done

17: Colony PCR will be repeated.

GEL GÖRÜNTÜSÜ

Colony PCR of “pColA blunt”

Colony PCR of “pColA blunt”
	MgCl₂	(NH₄)2SO₄	ColA fw	ColA rev	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
20X	50.0 ul	50.0 ul	20.0 ul	20.0 ul	10.0 ul	4.0 ul	246.0 ul		400.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: negative

GEL GÖRÜNTÜSÜ

PCR of “Q5 DNA Polymerase for pCAG” (19.08.2015)

PCR
	Template DNA	pCAG fw	pCAG rev	Master 2x Mix	ddH₂O	Total
1x	1.0 ul	5.0 ul	5.0 ul	25.0 ul	14.0 ul	50.0 ul

Cycling
	98˚C	98˚C	70˚C	72˚C	72˚C	Cycle
Time	30’’	10’’	30’’	1’	5’	35x

Result: Bands were at the expected section. PCR clean up will be made.

GEL GÖRÜNTÜSÜ

Digestion of “pCAG – Clean up”

Digestion
	pCAG – Clean up (34 ng/ul)	pCAG – Clean up (61.6 ng/ul)	EcoRI-HF (NEB)	XhoI (NEB)	Cut Smart Buffer	ddH₂O	Total
1	7.4 ul	-	0.5 ul	0.5 ul	2.0 ul	9.6 ul	20.0 ul
2	-	4.1 ul	0.5 ul	0.5 ul	2.0 ul	12.9 ul	20.0 ul

37˚C overnight

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/19/2015 7:08:39 PM	0.0	ng/ul	0.000	0.000	27.59	-0.07	DNA	50.00
2	pcag	biospec	8/19/2015 7:10:01 PM	61.6	ng/ul	1.233	0.686	1.80	1.02	DNA	50.00

Digestion of “pSB1C3 – mLacI miR373-BS” (19.08.2015)

Digestion
	mLacI miR373-BS	EcoRI-HF (NEB)	XhoI (NEB)	Cut Smart Buffer	ddH₂O	Total
1	3.4 ul	0.5 ul	0.5 ul	2.0 ul	13.9 ul	20.0 ul

37˚C 2.30h

Ligation of “pSB1C3 – mLacI miR373-BS with pTRE”

Ligation
	Digested mLacI miR373-BS with pTRE	pTRE	T4 DNA Ligase Buffer	T4 DNA Ligase	Cut Smart Buffer	ddH₂O	Total
1	3.0 ul	1.0 ul	2.0 ul	1.0 ul	2.0 ul	13.0 ul	20.0 ul

22˚C 1.30h

Transformation will be made.

Colony PCR of “#6,8” (20.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	ColA fw	ColA rev	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
25X	62.5 ul	62.5 ul	25.0 ul	25.0 ul	12.5 ul	5.0 ul	307.5 ul		500.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: Positive. Liquid Culture was made.

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/22/2015 12:11:34 PM	0.5	ng/ul	0.010	-0.003	-3.39	-3.90	DNA	50.00
2	8-1	biospec	8/22/2015 12:12:38 PM	70.3	ng/ul	1.406	0.755	1.86	1.64	DNA	50.00
3	8-3	biospec	8/22/2015 12:13:38 PM	80.7	ng/ul	1.613	0.846	1.91	2.11	DNA	50.00
4	6-1	biospec	8/22/2015 12:14:32 PM	33.3	ng/ul	0.666	0.354	1.88	1.44	DNA	50.00

Cut Check of “#6,8”

Cut Check
	DNA (400 ng/ul)	EcoRI	PstI	Fast Digest Buffer	ddH₂O	Total
1	5.7 ul	0.5 ul	0.5 ul	2.0 ul	11.3 ul	20.0 ul
2	4.8 ul	0.5 ul	0.5 ul	2.0 ul	12.2 ul	20.0 ul
3	12.0 ul	0.5 ul	0.5 ul	2.0 ul	5.0 ul	20.0 ul

37˚C 40’

Result: positive

GEL GÖRÜNTÜSÜ

Colony PCR “pSB1C3 17,6,8 – pColA, Damp-Pex, pTetoff” (19.08.2015)

GEL GÖRÜNTÜLERI

Mini Prep of “pSB1C3-#17”

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/20/2015 5:04:31 PM	0.6	ng/ul	0.013	0.007	1.93	1.40	DNA	50.00
2	psb1c3 17-2	biospec	8/20/2015 5:05:28 PM	78.9	ng/ul	1.579	0.821	1.92	2.17	DNA	50.00
3	psb1c3 17-5	biospec	8/20/2015 5:06:18 PM	77.5	ng/ul	1.551	0.806	1.92	2.08	DNA	50.00
4	psb1c3 17-8	biospec	8/20/2015 5:07:02 PM	87.5	ng/ul	1.749	0.910	1.92	2.08	DNA	50.00
5	psb1c3 17-10	biospec	8/20/2015 5:07:47 PM	105.4	ng/ul	2.108	1.087	1.94	2.15	DNA	50.00

Digestion of “pSB1C3 T7-10 and pET45 #4-1, #5-3, #6-6, #8-15” (21.08.2015)

Digestion pET45 #4-1, #5-3, #6-6, #8-15
	DNA	BamHI-HF (NEB)	XhoI (NEB)	Cut Smart Buffer	ddH₂O	Total
#4-1	10.1 ul	1.0 ul	1.0 ul	2.0 ul	5.9 ul	20.0 ul
#5-3	10.8 ul	1.0 ul	1.0 ul	2.0 ul	5.2 ul	20.0 ul
#6-6	5.0 ul	1.0 ul	1.0 ul	2.0 ul	11.0 ul	20.0 ul
#8-15	16.0 ul	1.0 ul	1.0 ul	2.0 ul	-	20.0 ul

37˚C 3h

Digestion pSB1C3 T7-10
	DNA	BamHI-HF (NEB)	XhoI (NEB)	Cut Smart Buffer	ddH₂O	Total
#4-1	4.8 ul	1.0 ul	1.0 ul	2.0 ul	11.2 ul	20.0 ul

1ul rSAP / 37˚C 1h

Ligation
	Vector (pSB1C3)	Insert	T4 DNA Ligase Buffer	T4 DNA Ligase	ddH₂O	Total
#4	1.0 ul	1.0 ul	2.0 ul	1.0 ul	15.0 ul	20.0 ul
#5	1.0 ul	1.0 ul	2.0 ul	1.0 ul	15.0 ul	20.0 ul
#6	1.0 ul	1.0 ul	2.0 ul	1.0 ul	15.0 ul	20.0 ul
#8	1.0 ul	1.0 ul	2.0 ul	1.0 ul	15.0 ul	20.0 ul

22˚C 2h

-80 ˚C 20’’ Heat Inaktivation

Colony PCR of “pTRE - #13 mLacI-miR373 BS” (21.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	CMV fw	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
22X	55.0 ul	55.0 ul	22.0 ul	22.0 ul	11.0 ul	4.4 ul	270.6 ul		440.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Results: negative

GEL GÖRÜNTÜLERI

Mini Prep of “pSB1C3 #6-25,26”

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/20/2015 10:01:33 AM	0.4	ng/ul	0.007	-0.003	-2.12	-1.13	DNA	50.00
2	psb1c3 #6-25	biospec	8/20/2015 10:02:45 AM	41.0	ng/ul	0.820	0.421	1.95	1.93	DNA	50.00
3	psb1c3 #6-26	biospec	8/20/2015 10:03:50 AM	46.0	ng/ul	0.920	0.464	1.98	1.97	DNA	50.00

Cut Check of “pSB1C3 #6-25,26”

Cut Check
	DNA	EcoRI-HF	PstI-HF	Cut Smart Buffer	ddH₂O	Total
#6-25	6.1 ul	0.5 ul	0.5 ul	2.0 ul	10.9 ul	20.0 ul
#6-26	5.5 ul	0.5 ul	0.5 ul	2.0 ul	11.5 ul	20.0 ul

37˚C 3h

Result: negative

Digestion of “pTRE #12”

Digestion
	pTRE #12 (5000 ng/ul)	EcoRI-HF	XhoI (NEB)	Cut Smart Buffer	ddH₂O	Total
1x	16.0 ul	0.5 ul	0.5 ul	2.0 ul	1.0 ul	20.0 ul

37˚C 3h

Result: negative

GEL GÖRÜNTÜSÜ

Digestion of “Damp-Pex pSB1C3” (22.08.2015)

Digestion pSB1C3 T7-10
	DNA (500 ng/ul)	NotI	Cut Smart Buffer	ddH₂O	Total
#9-5	3.42 ul	1.0 ul	2.0 ul	13.58 ul	20.0 ul
#9-10	3.56 ul	1.0 ul	2.0 ul	13.44 ul	20.0 ul

37˚C 3h

Ligation of “Damp-Pex pColA”

Ligation
	pColA (100 ng/ul)	Insert (36 ng/ul)	T4 DNA Ligase Buffer	T4 DNA Ligase	ddH₂O	Total
1x	2.8 ul	5.41 ul	2.0 ul	1.0 ul	15.0 ul	20.0 ul

22˚C 1.30h

Transformation with NEB5α.

Colony PCR of “Damp-Pex + pColA” (23.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	pColA fw	pColA rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
26X	65.0 ul	65.0 ul	26.0 ul	26.0 ul	13.0 ul	5.2 ul	319.8 ul		520.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: #5-4, #5-9 Liquid Culture was made.

GEL GÖRÜNTÜSÜ

Mini Prep of “pTetoff #14-4,5,8” (22.08.2015)

#	Sample ID	User name	Date and Time	Nucleic Acid Conc.	Unit	A260	A280	260/280	260/230	Sample Type	Factor
1	blank	biospec	8/22/2015 11:01:09 AM	0.1	ng/ul	0.002	-0.008	-0.23	-0.10	DNA	50.00
2	ptetoff 14-4	biospec	8/22/2015 11:02:05 AM	159.6	ng/ul	3.193	1.678	1.90	2.00	DNA	50.00
3	ptetoff 14-5	biospec	8/22/2015 11:03:21 AM	141.1	ng/ul	2.822	1.466	1.93	1.97	DNA	50.00
4	ptetoff 14-8	biospec	8/22/2015 11:04:14 AM	367.5	ng/ul	7.350	3.969	1.85	2.11	DNA	50.00

Cut Check I
	DNA (250 ng/ul)	SalI (FD)	HindIII (FD)	Fast Digest Buffer	ddH₂O	Total
#14-4	1.6 ul	0.5 ul	0.5 ul	2.0 ul	15.4 ul	20.0 ul
#14-5	1.8 ul	0.5 ul	0.5 ul	2.0 ul	15.2 ul	20.0 ul
#14-8	0.7 ul	0.5 ul	0.5 ul	2.0 ul	16.3 ul	20.0 ul

37˚C 2h

Result: negative

GEL GÖRÜNTÜSÜ

Cut Check II
	DNA (250 ng/ul)	SalI (FD)	NotI (FD)	Fast Digest Buffer	ddH₂O	Total
#14-4	1.6 ul	0.5 ul	0.5 ul	2.0 ul	15.4 ul	20.0 ul
#14-5	1.8 ul	0.5 ul	0.5 ul	2.0 ul	15.2 ul	20.0 ul
#14-8	0.7 ul	0.5 ul	0.5 ul	2.0 ul	16.3 ul	20.0 ul

37˚C 30’’

Result: negative

GEL GÖRÜNTÜSÜ

Colony PCR of “pSB1C3 – T7 #4,5,6,8” (23.08.2015)

Colony PCR
	MgCl₂	(NH₄)2SO₄	pTRE Luc fw	SV40 rv	dNTP	Tag	ddH₂O	DNA	Total
1x	2.5 ul	2.5 ul	1.0 ul	1.0 ul	0.5 ul	0.2 ul	12.3 ul	5.0 ul	25.0 ul
50x	125.0 ul	125.0 ul	50.0 ul	50.0 ul	25.0 ul	10.0 ul	615.0 ul		1000.0 ul

Cycling
	95˚C	95˚C	56˚C	72˚C	72˚C	Cycle
Time	5’	30’’	30’’	1.5’	5’	35x

Result: ????????

GEL GÖRÜNTÜSÜ

September

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

Week 1, 1.1.15-1.11.15

Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance..

Week 2, 1.12.15-1.18.15

Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance..

Week 3, 1.19.15-1.25.15

Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..

Week 4, 1.26.15-2.1.15

Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..