Team:Freiburg/Notebook Cloning

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Lab Notebook Cloning

April

19.4.17

Miniprep of pIG17_003/4

Miniprep was performed with QIAprep Spin Miniprep Kit.

Test Digestion of pIG17_003/4

Reaction conditions

pIG17_003 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
EcoRI-HF	20U/µl	1
CS-Buffer	10x	2
DNA	264 ng/µl	3

pIG17_004 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	15
EcoRI-HF	20U/µl	0.5
XbaI	20U/µl	0.5
CS-Buffer	10x	2
DNA	334 ng/µl	2

Electrophoresis failed and was repeated.

20.4.17

Miniprep of pIG17_001/2

Miniprep was performed with QIAprep Spin Miniprep Kit.

21.4.17

Test Digestion of pIG17_001/2/3/4

Reaction conditions

pIG17_001 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
EcoRI-HF	20U/µl	1
CS-Buffer	10x	2
DNA	178 ng/µl	3

pIG17_002 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	15
EcoRI-HF	20U/µl	0.5
XbaI	20U/µl	0.5
CS-Buffer	10x	2
DNA	246 ng/µl	2

pIG17_003 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
EcoRI-HF	20U/µl	1
CS-Buffer	10x	2
DNA	156ng/µl	3

pIG17_004 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
EcoRI-HF	20U/µl	1
CS-Buffer	10x	2
DNA	155ng/µl	3

22.4.17

Miniprep of pIG17_001/2

Miniprep was performed with QIAprep Spin Miniprep Kit.

Sequencing of pIG17_001/2

sequencing returned successful

23.4.17

Miniprep of pIG17_001/2

Miniprep was performed with QIAprep Spin Miniprep Kit.
Plasmids were used for PEI transfection.

25.4.17

Miniprep of pIG17_008/9

Miniprep was performed with QIAprep Spin Miniprep Kit.

DNA concentrations:
pIG17_008: 1043 ng/µl and 167,4 ng/µl
pIG17_009: 1295,1 ng/µl and 504,3 ng/µl

May

6.5.17

Test Digestion of pIG17_008/9

Reaction conditions

pIG17_008 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
ClaI	20U/µl	0,25
XbaI	20U/µl	0,25
CS-Buffer	10x	2
DNA	896ng/µl	0,5

pIG17_009 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
PvuII	20U/µl	0,5
CS-Buffer	10x	2
DNA	765ng/µl	0,5

PCR of pIG17_009

pIG17_009 Total reaction volume 25µl

component	concentration	volume [µL]
H20	-	15,75
oIG17_009 (Primer fw)	10µM	1,25
oIG17_010 (Primer rv)	10µM	1,25
5x Q5 Reaction Buffer	-	5
dNTPs	10mM	0,5
Polymerase Q5 HF	-	0,25
DNA	10ng/µl	1

7.5.17

Test Digestion of pIG17_008

Reaction conditions

pIG17_008 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
CalI	20U/µl	0,25
KpnI	20U/µl	0,25
CS-Buffer	10X	2
DNA	798ng/µl	0,5

pIG17_008 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
PstI	20U/µl	0,5
CS-Buffer	10x	2
DNA	886ng/µl	0,5

pIG17_008 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
XbaI	20U/µl	0,25
NdeI	20U/µl	0,25
CS-Buffer	-	2
DNA	µl	0,5

Gel Extraction of pIG17_009

Gel extraction was performed with QIAquick Gel Extraction Kit.

Concentration of pIG17_009: 51,5 ng/µl

9.5.17

Digestion of pIG17_002

Reaction conditions

pIG17_002 Total reaction volume 50µl

component	concentration	volume [µL]
H20	-	15
NdeI	20U/µl	1,5
AatII	20U/µl	1,5
CS-Buffer	10x	5
DNA	2µg	27

13.5.17

Gel Extraction of pIG17_002 --> SEAP and pA

Gel extraction was performed with QIAquick Gel Extraction Kit.

Concentration of pIG17_002: 84,5 ng/µl

Digestion of pIG17_001/002

Reaction conditions

pIG17_001 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13
NdeI	20U/µl	0,5
AatII	20U/µl	0,5
10x Fast Digest Green Buffer	-	2
DNA	500ng	4

undigested:

component	concentration	volume [µL]
H20	-	14
10x Fast Digest Green Buffer	-	2
DNA	500ng	4

pIG17_002 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13
NdeI	20U/µl	0,5
AatII	20U/µl	0,5
10x Fast Digest Green Buffer	-	2
DNA	500ng	4

undigested:

component	concentration	volume [µL]
H20	-	14
10x Fast Digest Green Buffer	-	2
DNA	500ng	4

PCR of pA/SEAP

pIG17_002 Total reaction volume 25µl

component	concentration	volume [µL]
H20	-	15,75
oIG17_012 (Primer fw)	10µM	1,25
oIG17_013 (Primer rv)	10µM	1,25
5x Q5 Reaction Buffer	-	5
dNTPs	10mM	0,5
Polymerase Q5 HF	-	0,25
DNA	10ng/µl	1

pIG17_002 Total reaction volume 25µl

component	concentration	volume [µL]
H20	-	15,75
oIG17_014 (Primer fw)	10µM	1,25
oIG17_015 (Primer rv)	10µM	1,25
5x Q5 Reaction Buffer	-	5
dNTPs	10mM	0,5
Polymerase Q5 HF	-	0,25
DNA	10ng/µl	1

Gel Ex of pA/SEAP

Gel extraction was performed with QIAquick Gel Extraction Kit.
Concentration of SEAP: 45,3 ng/µl
Concentration of pA: 49,1 ng/µl

15.5.17

Digestion of pIG17_002

Reaction conditions

pIG17_002 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	10
NdeI	20U/µl	1
AatII	20U/µl	1
CS-Buffer	10x	2
DNA	105,3 ng/µl	6

undigested: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	15,5
CS-Buffer	-	2
DNA	-	2,5

Because of the troubles to digest, the digestion was done for 2.5h by 37°C.

PCR of pIG17_009

pIG17_009 Total rection volume 25µl

component	concentration	volume [µL]
H20	-	15,75
oIG17_009 (Primer fw)	10µM	1,25
oIG17_010 (Primer rv)	10µM	1,25
5x Q5 Reaction Buffer	-	5
dNTPs	10mM	0,5
Polymerase Q5 HF	-	0,25
DNA	10ng/µl	1

18.5.17

Gibson Assembly of CMV, mCherry, pA and pSB1C3

pIG17_058 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,05pmol	2
CMV	0,05pmol	1
mCherry	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

Gibson Assembly of pIG17_012, pIG17_013, pIG17_015, pIG17_016

pIG17_012 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
HRE	0,05pmol	1
SEAP	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

pIG17_013 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
HRE	0,05pmol	1
CFP	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

pIG17_015 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
CRE	0,05pmol	1
SEAP	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

pIG17_016 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
CRE	0,05pmol	1
eCFP	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

PCR of pIG17_027/028/029

PCR of extracted genomic DNA of Jurkat cells to amplify CTLA4

pIG17_027/028/029 Total rection volume 25µl

component	concentration	volume [µL]
H20	-	8
oIG17_048/049/050 (Primer fw) (1000bp/380bp/330bp)	10µM	1,25
oIG17_051 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	2

Annealing temperatures:
CTLA4 1000bp: 66°C
CTLA4 380bp: 64°C
CTLA4 330bp: 65°C

Gel picture

CTLA4 380bp and 330bp have the expected length. For CTLA4 1000bp we couldn't see any band on the gel.

Gel Extraction of pIG17_028/029

CTLA4 1000bp was not extracted because it couldn't be seen on the gel.
Gel extraction was performed with QIAquick Gel Extraction Kit.

19.5.17

Gibson Assembly of CMV, mCherry delta PstI, pA and pSB1C3

pIG17_058 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,05pmol	2
CMV	0,05pmol	1
mCherry delta PstI	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

21.5.17

Subcloning of CTLA4_330/380

330 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pJET2.1	50ng/µl	1
DNA	5ng/µl	5

380 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pJET2.1	50ng/µl	1
DNA	5ng/µl	5

Sequencing of pIG17_012/13/15/16

Sequencing returned positive

22.5.17

Test digestion of mCherry and the Gibson Assembly G1.1

Digestion of the constructs was performed to test the function of the enzymes.

Reaction conditions

mCherry Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
KpnI	20U/µl	0,25
CS-Buffer	10x	2
DNA	-	4

undigested: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
CS-Buffer	-	2
DNA	-	4

undigested without CS-Buffer: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	16
DNA	-	4

Gibson Assembly G1.1 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	12,25
HindIII	20U/µl	0,25
AflII	20U/µl	0,25
ClaI	20U/µl	0,25
CS-Buffer	10x	2
DNA	-	5

undigested: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13
CS-Buffer	-	2
DNA	-	5

Gel picture

Because of the lack of an AafII restriction site in the G1.1 construct there are only two bands to be seen.
All bands have the expected length and the enzymes still work.

23.5.17

Test digestion of CTLA4 380bp and 330bp

Reaction conditions

CTLA4 330 (3,4) and CTLA4 380 (1) Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	12,5
NotI	20U/µl	0,25
NcoI-HF	20U/µl	0,25
CS-Buffer	-	2
DNA	500ng	5

CTLA4 330 (1,2) and CTLA4 380 (3,4) Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,5
NotI	20U/µl	0,25
NcoI-HF	20U/µl	0,25
CS-Buffer	-	2
DNA	500ng	4

CTLA4 380 (2) Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14,5
NotI	20U/µl	0,25
NcoI-HF	20U/µl	0,25
CS-Buffer	-	2
DNA	500ng	3

Undigested samples were done, respectively.

Gel picture

CTLA4 330bp number 1 and also number 1 of CTLA4 380bp were chosen for further procedures.

30.5.17

Obtaining pSAM200 and pMF111 form AG Weber

31.5.17

Test digestion of pSAM200/pMF111

pSAM200 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,5
XbaI	20U/µl	0,25
XhoI	20U/µl	0,25
CS-Buffer	10x	2
DNA	170,3ng/µl	4

pMF200 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,5
XbaI	20U/µl	0,25
XhoI	20U/µl	0,25
CS-Buffer	10x	2
DNA	133,7ng/µl	4

June

3.6.17

Midiprep of pIG17_003/082

Midiprep was performed with the Promega PureYield Plasmid Midiprep System Kit.

Miniprep of CTLA4 (330bp, 380bp)

Miniprep was performed with QIAprep Spin Miniprep Kit.

Extension PCR of CTLA4 (330bp, 380bp)

CTLA4 (330bp)
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_105 (Primer fw)	10µM	1,25
oIG17_106 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

CTLA4 (380bp)
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_107 (Primer fw)	10µM	1,25
oIG17_106 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

Annealing temperatures:
CTLA4 380bp: 59°C
CTLA4 330bp: 65°C

4.6.17

Extension PCR of CTLA4 (330bp, 380bp)

Because the extension PCR didn't work the day before, we performed another one.

CTLA4 (330bp)
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_105 (Primer fw)	10µM	1,25
oIG17_106 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

CTLA4 (380bp)
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_107 (Primer fw)	10µM	1,25
oIG17_106 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

Annealing temperatures:
CTLA4 380bp: 59°C
CTLA4 330bp: 65°C

Gel Extraction of CTLA4 (330bp, 380bp) of the extension PCR

Gel extraction was performed with QIAquick Gel Extraction Kit.

Gibson Assembly of CTLA4, GFP/SEAP, pA and BB - pIG17_019/020/022/023

CTLA4 (330bp) and GFP - pIG17_023
Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,06pmol	0,75
CTLA4 (330bp)	0,06pmol	1
GFP	0,06pmol	0,5
pA	0,06pmol	1
H2O	-	1,75

CTLA4 (330bp) and SEAP - pIG17_020
Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,06pmol	0,75
CTLA4 (330bp)	0,06pmol	1
SEAP	0,06pmol	0,75
pA	0,06pmol	1
H2O	-	1,5

CTLA4 (380bp) and GFP - pIG17_022
Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,06pmol	0,75
CTLA4 (380bp)	0,06pmol	0,5
GFP	0,06pmol	0,5
pA	0,06pmol	1
H2O	-	2,25

CTLA4 (380bp) and SEAP - pIG17_019
Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,06pmol	0,75
CTLA4 (380bp)	0,06pmol	0,5
SEAP	0,06pmol	0,75
pA	0,06pmol	1
H2O	-	2

Placed at 50°C for 1h

Transformation pIG17_019/020/022/023/030

Transformation was done with DH5alpha Z-competent cells for all CTLA4-GFP/SEAP Gibson assemblies.
pIG17_030 was done again because there were no colonies on the dish.

5.6.17

Midiprep of pIG17_004/008/009

Midiprep was performed with the Promega PureYield Plasmid Midiprep System Kit.

Gel

Because of the trouble to get a transformation of pIG17_030, the Gibson assembly and the used constructs were loaded on a gel, in order to find out where the problem lies.

Result of Gibson assembly

CTLA4 330 with GFP did growth
CTLA4 380 with GFP did growth
CTLA4 330 with SEAP did not growth
CTLA4 380 with SEAP did not growth

6.6.17

Extention PCR of LUC

Extention PCR made to get rid of unwanted the cutting site

Miniprep of CTLA4_GFP (pIG17.022, pIG17.023)

Miniprep with JenaBioscience. The yield was too poor to work with. New cultures are needed. o/n are done.

Sequencing of SEAP

SEAP construct (19, 20) are send to sequencing.

Sequencing analysis of pCRE and pHRE (pIG17.006, pIG17.007)

The results:

Pick of CTLA4 330_SEAP (pIG17.020)

Test digest of CTLA4 330 and 380 SEAP & GFP (pIG17.019, pIG17.020, pIG17.022, pIG17.023)

Construct		Enzym	Size	Size undigested
CTLA4 330	GFP	HindI II	2210bp, 925bp	3127pb
	SEAP	HindI II	2210pb, 1726pb	3928pb
CTLA4 380	GFP	HindI II	2260pb, 929pb	3177pb
	SEAP	HindI II	2260pb, 1726pb	3978pb

Transformation of HRE_SEAP (pIG17.030)

Transformation made with 5μl. Did not worked.

Test digest of Luceferase

Concentration: 462ng/μl

	Volumina for digest	Volumina for negative control
DNA	1,1μl	1,1μl
Buffer	2μl	2μl
Water	16,4μl	16,9μl
Enzyme 1	0,25μl
Enzyme 2	0,25μl
Total	20μl	20μl

Used enzyme: 1:XbaI: 37°C, 2:NotI:37°C Buffer: NEBuffer 3.1 DNA used: 500ng

Test digest of pIG17.019, pIG17.020, pIG17.022, pIG17.023

	330 GFP	330 SEAP	380 GFP	330 SEAP	380 SEAP
DNA	250ng	250ng	250ng	250ng	250ng
Water
Enzyme	0,5μl	0,5μl	0,5μl	0,5μl	0,5μl
Buffer	2μl	2μl	2μl	2μl	2μl
Total	20μl	20μl	20μl	20μl	20μl

On the gel: Loading dye: 5μl

Midi Prep of CMV (pIG17.008, pIG17.009)

Midiprep done with Promega PureYield™ Plasmid Midiprep System kit

	1.elution	2.elution
pIG17.008	142,1ng/μl	50,1ng/μl
pIG17.009	89,3ng/μl	64,1ng/μl

7.6.17

Miniprep of pIG17_019/020/022/023

Miniprep was performed with Jena Bioscience Kit.

Results

Transformation of HRE SEAP with mCherry didn't grow

Test digestof pIG17_019/020/022/023

Test diget with HindIII
DNA concentration of 500ng/μl (4μl of DNA) 10xPuffer (2μl) H2O till 20μl in the PCR tube.

GATC sequencing

3x HIF1-a with 30ng/μl with the primer oIG17_119 (3μl with 26μl H2O)

8.6.17

Trouble shooting of the Promega Midiprep kit

Extention PCR with mCherry and pIG17_012

9.6.17

Test Digestion of pIG17_019/020/022/023

Reaction conditions

pIG17_019/020: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17,5 - x
Nde I	20U/µl	0,25
Cla I	20U/µl	0,25
CS-Buffer	-	2
DNA	500 ng	x

pIG17_022/023: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17,5 - x
Nco I	20U/µl	0,25
Cla I	20U/µl	0,25
CS-Buffer	-	2
DNA	500 ng	x

Undigested:
Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	18 - x
CS-Buffer	-	2
DNA	500 ng	x

Incubation for 1h at 37 degrees

Gel extraction of pIG17_012 and mCherry

DNA	Gel	QG	Isopropanol
pIG17_012	0,12g	240μl	81,16
mCherry	0,08g	360μl	122,4μl

Midiprep of pIG17_008 and pIG17_009

2 bands on the gel : Contamination!

14.6.17

Test Digestion of SDM of LUC

SDM of LUC Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
Tango Buffer	10x	2
DNA	151,7ng/µl	4

Gibson assembly pIG17_31/34/37/86

pIG17_31 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
pIG17_012	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_34 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
pIG17_013	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_37 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
pIG17_015	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_86 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
pIG17_016	0,05pmol	1
pIG17_058	0,05pmol	1

12.6.17

Midiprep of pIG17_008/009

Midiprep was performed with the Promega Kit.

Gibson Assembly of Luciferase with SDM

Total rection volume 24µl

component	concentration	volume [µL]
2x Gibson MM	-	12
Luciferase Backbone	0,06pmol	7,5
PCR product 1 of Luciferase	0,06pmol	1
PCR product 2 of Luciferase	0,06pmol	2,5
PCR product 3 of Luciferase	0,05pmol	0,5
Water	-	0,5

Placed at 50°C for 1h

6.7.17

Mini Prep of HIF1 - alpha fusion proteins (pmf111/psb1c3/LUC delta BspEI)

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pmf111	48,1ng/μl
psb1c3	107,6ng/μl
LUC delta BspEI	289,6ng/μl

Miniprep of pIG17_003/4

Miniprep was not performed due to the fact that the overnight culture got spilled out.

PCR of HIF1 - alpha fusion proteins (psb1c3/LUC delta BspEI)

psb1c3
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_198 (Primer fw)	10µM	1,25
oIG17_197 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

LUC delta BspEI
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_101 (Primer fw)	10µM	1,25
oIG17_202 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

LUC delta BspEI
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_203 (Primer fw)	10µM	1,25
oIG17_100 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

17.6.17

Testdigestion of pIG17_31/34/37/86

pIG17_031 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	120,7ng/µl	4

pIG17_034 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	130,5ng/µl	4

pIG17_037 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	111,7ng/µl	4

pIG17_087 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	101,9ng/µl	4

Positive clones were send to sequencing

18.6.17

Gibson Assembly of pIG17_063/064/065

pIG17_063 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
HRE	0,05pmol	1
LUC	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_064 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
CRE	0,05pmol	1
LUC	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_065 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
NFAT b.s.	0,05pmol	1
LUC	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

22.06.17

Test digestion of pIG17_063/064/065

pIG17_063 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	100,7ng/µl	4

pIG17_064 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	120,5ng/µl	4

pIG17_064 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	98,1ng/µl	4

Positive clones were sent to sequencing

28.6.17

Digestion of pIG17_31/34/37/86 for ligation into Transferplasmid

pIG17_031 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
ClaI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	120,1ng/µl	4

pIG17_034 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
ClaI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	160,8ng/µl	4

pIG17_037 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
ClaI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	143,3ng/µl	4

pIG17_086 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
ClaI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	198,5ng/µl	4

Transferplasmid Total reaction volume 50µl

component	concentration	volume [µL]
H20	-	30,25
BspEI	20U/µl	0,5
KpnI	20U/µl	0,5
Tango Buffer	10x	2
DNA	300,4ng/µl	4

T4 Ligation of pIG17_31/34/37/87 into Transfer plasmid

pIG17_31 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
Transfer plasmid	70ng/µl	1
pIG17_031	13ng/µl	5

pIG17_34 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
Transfer plasmid	70ng/µl	1
pIG17_034	15ng/µl	5

pIG17_37 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
Transfer plasmid	70ng/µl	1
pIG17_037	13ng/µl	5

pIG17_87 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
Transfer plasmid	70 ng/µl	1
pIG17_087	5ng/µl	5

July

2.7.17

Gibson Assembly of HRE-GFP-pA-SV40-Neomycin/Zeozin

HRE-GFP-pA-SV40-Neomycin Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
pIg17_013	0,05pmol	1
SV40	0,05pmol	1
Neomycin	0,05pmol	1
pA	0,05pmol	1

HRE-GFP-pA-SV40-Zeozinin Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
pIg17_013	0,05pmol	1
SV40	0,05pmol	1
Zeocin	0,05pmol	1
pA	0,05pmol	1

03.07.17

Gibson assembly of SV40-TetR-pA

HRE-GFP-pA-SV40-Neomycin Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
SV40	0,05pmol	1
VP16-TetR	0,05pmol	1
pA	0,05pmol	1

7.7.17

Mini Prep of pIG17_031/034/086/119

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pIG17.031	46,6ng/μl
pIG17.034	45,7ng/μl
pIG17.086	47,9ng/μl
pIG17.119	390,8ng/μl

PCR of HIF1 - alpha fusion proteins (pMF111/pSB1C3(standard part we got from iGEM))

pMF111 #1
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_177 (Primer fw)	10µM	1,25
oIG17_211 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

pMF111 #3
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_150 (Primer fw)	10µM	1,25
oIG17_176 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

pSB1C3 #2
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_198 (Primer fw)	10µM	1,25
oIG17_197 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

Gel picture

Midiprep of 31, 34, 86, 119

Midiprep with Promega Midiprep kit

pIG17_031	6,0ng/μl
pIG17_034	4,4ng/μl
pIG17_086	9,4ng/μl	6,9ng/μl
pIG17_119	156ng/μl	167ng/μl

labbooks

8.7.17

Gibson Assembly of TetO/HRE/CRE/NFAT b.s.-HIF/HIF-LUC-CMV-mCherry

TetO-HIF-LUC-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	6
pcDNA3.1	0,05pmol	1
TetO	0,05pmol	1
HIF	0,05pmol	1
LUC	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

HRE-HIF-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
HRE	0,05pmol	1
HIF	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

CRE-HIF-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
CRE	0,05pmol	1
HIF	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

NFAT bs 330-HIF-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
NFAT b.s. 330	0,05pmol	1
HIF	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

NFAT bs 380-HIF-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
NFAT b.s. 380	0,05pmol	1
HIF	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

9.7.17

Bacterial Glycerol Stocks for Long-term Storage of Plasmids

• pIG17_037
• pIG17_086
• pIG17_119

Mini Prep of pIG17_118/120/121

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pIG17_118	51,6ng/μl
pIG17_120 #1	50ng/μl
pIG17_120 #2	56ng/μl
pIG17_121 #1	208,3ng/μl
pIG17_121 #2	56ng/μl

10.7.17

Mini Prep of pIG17_31 zeomycin and neomycin/pIG17_097

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pIG17_031 zeo	107,9ng/μl
pIG17_031 neo	25,7ng/μl
pIG17_097 1 #1	30,8ng/μl
pIG17_097 1 #2	34,2ng/μl
pIG17_097 2 #1	38,4ng/μl
pIG17_097 2 #2	40,0ng/μl
pIG17_097 2 #3	32,3ng/μl

12.7.17

Mini Prep of TetO-Luc-HIF/TetO-HIF-Luc/pIG17_031 TetR

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pIG17_031 TetR	22,0ng/μl
TetO-Luc-HIF #1	32,4ng/μl
TetO-HIF-Luc #1	174,5ng/μl

13.7.17

Mini Prep of pCRE-HIF/CTLA4-HIF

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pCRE-HIF #3	28,3ng/μl
pCRE-HIF #2	30,1ng/μl
pCRE-HIF #1	30,4ng/μl
CTLA4-HIF(380) #1	32,1ng/μl
CTLA4-HIF(380) #2	33,1ng/μl
CTLA4-HIF(380) #3	32,8ng/μl
CTLA4-HIF(330) #2	29,9ng/μl

15.7.17

Ligation of HRE*4/CRE*4/NFAT bs*4 into pIG17_031

HRE*4 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	6
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pIG17_031	65ng/µl	1
HRE*4	10ng/µl	1

CRE*4 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	6
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pIG17_031	65ng/µl	1
CRE*4	10ng/µl	1

NFAT bs*4 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	6
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pIG17_031	65ng/µl	1
NFAT bs*4	10ng/µl	1

16.7.17

Gibson Assembly of HRE*4/CRE*4/NFAT bs*4-CFP-CMV-mCherry

HRE*4-CFP-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
Transfer vector	0,05pmol	2
HRE*4	0,05pmol	1
CFP	0,05pmol	1
pIG17_058	0,05pmol	1

CRE*4-CFP-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
Transfer vector	0,05pmol	2
CRE*4	0,05pmol	1
CFP	0,05pmol	1
pIG17_058	0,05pmol	1

NFAT bs*4-CFP-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
Transfer vector	0,05pmol	2
NFAT bs*4	0,05pmol	1
CFP	0,05pmol	1
pIG17_058	0,05pmol	1

19.7.17

Mini Prep of pCRE-HIF/CTLA4-HIF/pIG17_096

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pCRE-HIF	26,7ng/μl
pCRE-HIF	187,7ng/μl
pCRE-HIF	153,0ng/μl
pCRE-HIF	102,0ng/μl
CTLA4-HIF(380)	32,4ng/μl
CTLA4-HIF(380)	340,1ng/μl
CTLA4-HIF(380)	273,5ng/μl
CTLA4-HIF(380)	70,4ng/μl
CTLA4-HIF(380)	88,81ng/μl
CTLA4-HIF(380)	59,9ng/μl
CTLA4-HIF(330)	29,0ng/μl
CTLA4-HIF(330)	315,2ng/μl
CTLA4-HIF(330)	98,8ng/μl
CTLA4-HIF(330)	344,6ng/μl
CTLA4-HIF(330)	135,1ng/μl
pIG17_096	342,8ng/μl
pIG17_096	31,3ng/μl
pIG17_096	71,3ng/μl
pIG17_096	99,7ng/μl
pIG17_096	55,8ng/μl

Sequencing of pCRE-HIF/CTLA4-HIF 330/CTLA4-HIF 330/pIG17_096/pIG17_097/pIG17_118/pIG17_120

plasmid number	primer
pCRE-HIF	oIG17_166/018/164/174/175/119
CTLA4-HIF(380)	oIG17_166/018/164/174/175/119
CTLA4-HIF(330)	oIG17_166/018/164/174/175/119
pIG17_096	oIG17_174/175/169/170/018
pIG17_097	oIG17_054
pIG17_118	oIG17_019
pIG17_120	oIG17_019

21.7.17

Mini Prep of HRE x Mult-GFP-CMV-mCherry/CRE x Mult-GFP-CMV-mCherry/HRE ecFP zeo/TetR VP16 neo

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration	curve
HRE x Mult-GFP-CMV-mCherry #1	39,4ng/μl
HRE x Mult-GFP-CMV-mCherry #2	28,9ng/μl
HRE x Mult-GFP-CMV-mCherry #3	28,9ng/μl
HRE x Mult-GFP-CMV-mCherry #4	44,6ng/μl
CRE x Mult-GFP-CMV-mCherry #1	18,9ng/μl
CRE x Mult-GFP-CMV-mCherry #2	49,8ng/μl
CRE x Mult-GFP-CMV-mCherry #3	20,9ng/μl
CRE x Mult-GFP-CMV-mCherry #4	25,2ng/μl
HRE ecFP zeo #1	35,2ng/μl
HRE ecFP zeo #2	26,4ng/μl
HRE ecFP zeo #3	36,2ng/μl
HRE ecFP zeo #4	17,3ng/μl
TetR VP16 neo #1	21,7ng/μl
TetR VP16 neo #2	30,4ng/μl
TetR VP16 neo #3	66,7ng/μl
TetR VP16 neo #4	28,1ng/μl

• we also went upstairs to check our concentrations with Toolbox Nanodrop but there wasn't much difference
• Denis asked Nicole if she can do a Miniprep for us with her kit
• Nicole suggested to try to use the buffer instead of water to see if it will change something

24.7.17

Mini Prep of pIG17_012/013/015/016

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration	curve
pIG17_012	187,1ng/μl
pIG17_013	53,9ng/μl
pIG17_015	49,1ng/μl
pIG17_016	26,1ng/μl

29.7.17

Mini Prep of pIG17_003/031/119/

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit (in 40μl Wasser eluiert)

	concentration
pIG17_003	16,9ng/μl
pIG17_003	90,8ng/μl
pIG17_003	144,0ng/μl
pIG17_004	154,9ng/μl
pIG17_004	280,7ng/μl
pIG17_031	541,1ng/μl (!)
pIG17_031	257,1ng/μl (!)
pIG17_031	336,1ng/μl (!)
pIG17_034	23,0ng/μl (!)
pIG17_119	318,6ng/μl (!)
pIG17_119	19,3ng/μl
pIG17_119	31,9ng/μl
pIG17_119	12,1ng/μl
pIG17_119	53,2ng/μl
pIG17_119	314,0ng/μl (!)
HRE GFP	109,9ng/μl
HRE GFP	43,9ng/μl
HRE GFP	89,9ng/μl
HRE GFP	147,1ng/μl
HRE GFP	104,3ng/μl
HRE GFP	67,9ng/μl (!)
HRE GFP	125,2ng/μl
HRE GFP	153,1ng/μl
HRE GFP	318,2ng/μl
HRE GFP	294,9ng/μl
HRE GFP	148,6ng/μl
HRE GFP	82,1ng/μl
HRE GFP	423,9ng/μl (!)
HRE GFP	427,2ng/μl (!)

30.7.17

Mini Prep of pIG17_503/Tet-GFP

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit (in 40μl Wasser eluiert)

	concentration	curve
pIG17_503	242,6ng/μl
Tet-GFP	255,1ng/μl