Team:ATOMS-Turkiye/Extras/Notebook

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Team ATOMS-Turkiye

atoms2015@googlegroups.com

NOTEBOOK

December

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January

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February

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March

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April

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May

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June

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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, 1.19.15-1.25.15

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

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..

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, 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..

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..