Team:Freiburg/Notebook Cloning

labor:cell_culture - iGEM 2017

Lab Notebook Cloning

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

componentconcentrationvolume [µL]
H20-14
EcoRI-HF20U/µl1
CS-Buffer10x2
DNA264 ng/µl3

pIG17_004 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-15
EcoRI-HF20U/µl0.5
XbaI20U/µl0.5
CS-Buffer10x2
DNA334 ng/µl2

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

componentconcentrationvolume [µL]
H20-14
EcoRI-HF20U/µl1
CS-Buffer10x2
DNA178 ng/µl3

pIG17_002 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-15
EcoRI-HF20U/µl0.5
XbaI20U/µl0.5
CS-Buffer10x2
DNA246 ng/µl2

pIG17_003 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-14
EcoRI-HF20U/µl1
CS-Buffer10x2
DNA156ng/µl3

pIG17_004 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-14
EcoRI-HF20U/µl1
CS-Buffer10x2
DNA155ng/µl3

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

6.5.17

Test Digestion of pIG17_008/9

Reaction conditions

pIG17_008 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-17
ClaI20U/µl0,25
XbaI20U/µl0,25
CS-Buffer10x2
DNA896ng/µl0,5

pIG17_009 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-17
PvuII20U/µl0,5
CS-Buffer10x2
DNA765ng/µl0,5

PCR of pIG17_009

pIG17_009 Total reaction volume 25µl

componentconcentrationvolume [µL]
H20-15,75
oIG17_009 (Primer fw)10µM1,25
oIG17_010 (Primer rv)10µM1,25
5x Q5 Reaction Buffer-5
dNTPs10mM0,5
Polymerase Q5 HF-0,25
DNA10ng/µl1

7.5.17

Test Digestion of pIG17_008

Reaction conditions

pIG17_008 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-17
CalI20U/µl0,25
KpnI20U/µl0,25
CS-Buffer10X2
DNA798ng/µl0,5

pIG17_008 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-17
PstI20U/µl0,5
CS-Buffer10x2
DNA886ng/µl0,5

pIG17_008 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-17
XbaI20U/µl0,25
NdeI20U/µl0,25
CS-Buffer-2
DNAµl0,5

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

componentconcentrationvolume [µL]
H20-15
NdeI20U/µl1,5
AatII20U/µl1,5
CS-Buffer10x5
DNA2µg27

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

componentconcentrationvolume [µL]
H20-13
NdeI20U/µl0,5
AatII20U/µl0,5
10x Fast Digest Green Buffer-2
DNA500ng4

undigested:

componentconcentrationvolume [µL]
H20-14
10x Fast Digest Green Buffer-2
DNA500ng4

pIG17_002 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13
NdeI20U/µl0,5
AatII20U/µl0,5
10x Fast Digest Green Buffer-2
DNA500ng4

undigested:

componentconcentrationvolume [µL]
H20-14
10x Fast Digest Green Buffer-2
DNA500ng4

PCR of pA/SEAP

pIG17_002 Total reaction volume 25µl

componentconcentrationvolume [µL]
H20-15,75
oIG17_012 (Primer fw)10µM1,25
oIG17_013 (Primer rv)10µM1,25
5x Q5 Reaction Buffer-5
dNTPs10mM0,5
Polymerase Q5 HF-0,25
DNA10ng/µl1

pIG17_002 Total reaction volume 25µl

componentconcentrationvolume [µL]
H20-15,75
oIG17_014 (Primer fw)10µM1,25
oIG17_015 (Primer rv)10µM1,25
5x Q5 Reaction Buffer-5
dNTPs10mM0,5
Polymerase Q5 HF-0,25
DNA10ng/µl1

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

componentconcentrationvolume [µL]
H20-10
NdeI20U/µl1
AatII20U/µl1
CS-Buffer10x2
DNA105,3 ng/µl6

undigested: Total reaction volume 20µl

componentconcentrationvolume [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

componentconcentrationvolume [µL]
H20-15,75
oIG17_009 (Primer fw)10µM1,25
oIG17_010 (Primer rv)10µM1,25
5x Q5 Reaction Buffer-5
dNTPs10mM0,5
Polymerase Q5 HF-0,25
DNA10ng/µl1

18.5.17

Gibson Assembly of CMV, mCherry, pA and pSB1C3

pIG17_058 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pSB1C3 Backbone0,05pmol2
CMV0,05pmol1
mCherry0,05pmol1
pA0,05pmol1

Placed at 50°C for 1h

Gibson Assembly of pIG17_012, pIG17_013, pIG17_015, pIG17_016

pIG17_012 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
HRE0,05pmol1
SEAP0,05pmol1
pA0,05pmol1

Placed at 50°C for 1h

pIG17_013 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
HRE0,05pmol1
CFP0,05pmol1
pA0,05pmol1

Placed at 50°C for 1h

pIG17_015 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
CRE0,05pmol1
SEAP0,05pmol1
pA0,05pmol1

Placed at 50°C for 1h

pIG17_016 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
CRE0,05pmol1
eCFP0,05pmol1
pA0,05pmol1

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

componentconcentrationvolume [µL]
H20-8
oIG17_048/049/050 (Primer fw) (1000bp/380bp/330bp)10µM1,25
oIG17_051 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl2

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pSB1C3 Backbone0,05pmol2
CMV0,05pmol1
mCherry delta PstI0,05pmol1
pA0,05pmol1

Placed at 50°C for 1h

21.5.17

Subcloning of CTLA4_330/380

330 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-2
T4 Ligase -2
T4 Ligase Reaction buffer2x10
pJET2.150ng/µl1
DNA5ng/µl5

380 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-2
T4 Ligase -2
T4 Ligase Reaction buffer2x10
pJET2.150ng/µl1
DNA5ng/µl5

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

componentconcentrationvolume [µL]
H20-13,75
KpnI20U/µl0,25
CS-Buffer10x2
DNA-4

undigested: Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-14
CS-Buffer-2
DNA-4

undigested without CS-Buffer: Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-16
DNA-4

Gibson Assembly G1.1 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-12,25
HindIII20U/µl0,25
AflII20U/µl0,25
ClaI20U/µl0,25
CS-Buffer10x2
DNA-5

undigested: Total reaction volume 20µl

componentconcentrationvolume [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

componentconcentrationvolume [µL]
H20-12,5
NotI20U/µl0,25
NcoI-HF20U/µl0,25
CS-Buffer-2
DNA500ng5

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

componentconcentrationvolume [µL]
H20-13,5
NotI20U/µl0,25
NcoI-HF20U/µl0,25
CS-Buffer-2
DNA500ng4

CTLA4 380 (2) Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-14,5
NotI20U/µl0,25
NcoI-HF20U/µl0,25
CS-Buffer-2
DNA500ng3

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

componentconcentrationvolume [µL]
H20-13,5
XbaI20U/µl0,25
XhoI20U/µl0,25
CS-Buffer10x2
DNA170,3ng/µl4

pMF200 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,5
XbaI20U/µl0,25
XhoI20U/µl0,25
CS-Buffer10x2
DNA133,7ng/µl4

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

componentconcentrationvolume [µL]
H20-9
oIG17_105 (Primer fw)10µM1,25
oIG17_106 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

CTLA4 (380bp)
Total rection volume 25µl

componentconcentrationvolume [µL]
H20-9
oIG17_107 (Primer fw)10µM1,25
oIG17_106 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

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

componentconcentrationvolume [µL]
H20-9
oIG17_105 (Primer fw)10µM1,25
oIG17_106 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

CTLA4 (380bp)
Total rection volume 25µl

componentconcentrationvolume [µL]
H20-9
oIG17_107 (Primer fw)10µM1,25
oIG17_106 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pSB1C3 Backbone0,06pmol0,75
CTLA4 (330bp)0,06pmol1
GFP0,06pmol0,5
pA0,06pmol1
H2O-1,75

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pSB1C3 Backbone0,06pmol0,75
CTLA4 (330bp)0,06pmol1
SEAP0,06pmol0,75
pA0,06pmol1
H2O-1,5

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pSB1C3 Backbone0,06pmol0,75
CTLA4 (380bp)0,06pmol0,5
GFP0,06pmol0,5
pA0,06pmol1
H2O-2,25

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pSB1C3 Backbone0,06pmol0,75
CTLA4 (380bp)0,06pmol0,5
SEAP0,06pmol0,75
pA0,06pmol1
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, 1726pb3928pb
CTLA4 380 GFP HindI II2260pb, 929pb 3177pb
SEAP HindI II 2260pb, 1726pb3978pb

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
DNA1,1μl 1,1μl
Buffer2μl 2μl
Water16,4μl16,9μl
Enzyme 10,25μl
Enzyme 20,25μl
Total20μl 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 GFP330 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
Total20μl20μl20μl20μl20μl

On the gel: Loading dye: 5μl

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

Midiprep done with Promega PureYield™ Plasmid Midiprep System kit

1.elution2.elution
pIG17.008142,1ng/μl50,1ng/μl
pIG17.00989,3ng/μl64,1ng/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

componentconcentrationvolume [µL]
H20-17,5 - x
Nde I20U/µl0,25
Cla I20U/µl0,25
CS-Buffer-2
DNA500 ngx


pIG17_022/023: Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-17,5 - x
Nco I20U/µl0,25
Cla I20U/µl0,25
CS-Buffer-2
DNA500 ngx

Undigested:
Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-18 - x
CS-Buffer-2
DNA500 ngx


Incubation for 1h at 37 degrees

Gel extraction of pIG17_012 and mCherry

DNAGelQGIsopropanol
pIG17_0120,12g240μl81,16
mCherry0,08g360μl122,4μl

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

componentconcentrationvolume [µL]
H20-13,75
BspEI20U/µl0,25
Tango Buffer10x2
DNA151,7ng/µl4

Gibson assembly pIG17_31/34/37/86

pIG17_31 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
pIG17_0120,05pmol1
pIG17_0580,05pmol1

pIG17_34 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
pIG17_0130,05pmol1
pIG17_0580,05pmol1

pIG17_37 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
pIG17_0150,05pmol1
pIG17_0580,05pmol1

pIG17_86 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
pIG17_0160,05pmol1
pIG17_0580,05pmol1

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

componentconcentrationvolume [µL]
2x Gibson MM-12
Luciferase Backbone0,06pmol7,5
PCR product 1 of Luciferase0,06pmol1
PCR product 2 of Luciferase0,06pmol2,5
PCR product 3 of Luciferase0,05pmol0,5
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
pmf11148,1ng/μl
psb1c3107,6ng/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

componentconcentrationvolume [µL]
H20-9
oIG17_198 (Primer fw)10µM1,25
oIG17_197 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

LUC delta BspEI
Total rection volume 25µl

componentconcentrationvolume [µL]
H20-9
oIG17_101 (Primer fw)10µM1,25
oIG17_202 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

LUC delta BspEI
Total rection volume 25µl

componentconcentrationvolume [µL]
H20-9
oIG17_203 (Primer fw)10µM1,25
oIG17_100 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

17.6.17

Testdigestion of pIG17_31/34/37/86

pIG17_031 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
BspEI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA120,7ng/µl4

pIG17_034 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
BspEI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA130,5ng/µl4

pIG17_037 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
BspEI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA111,7ng/µl4

pIG17_087 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
BspEI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA101,9ng/µl4

Positive clones were send to sequencing

18.6.17

Gibson Assembly of pIG17_063/064/065

pIG17_063 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
HRE0,05pmol1
LUC0,05pmol1
pA0,05pmol1
pIG17_0580,05pmol1

pIG17_064 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
CRE0,05pmol1
LUC0,05pmol1
pA0,05pmol1
pIG17_0580,05pmol1

pIG17_065 Total rection volume 10µl

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
NFAT b.s.0,05pmol1
LUC0,05pmol1
pA0,05pmol1
pIG17_0580,05pmol1

22.06.17

Test digestion of pIG17_063/064/065

pIG17_063 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
BspEI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA100,7ng/µl4

pIG17_064 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
BspEI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA120,5ng/µl4

pIG17_064 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
BspEI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA98,1ng/µl4

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

componentconcentrationvolume [µL]
H20-13,75
ClaI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA120,1ng/µl4

pIG17_034 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
ClaI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA160,8ng/µl4

pIG17_037 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
ClaI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA143,3ng/µl4

pIG17_086 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-13,75
ClaI20U/µl0,25
KpnI20U/µl0,25
Tango Buffer10x2
DNA198,5ng/µl4

Transferplasmid Total reaction volume 50µl

componentconcentrationvolume [µL]
H20-30,25
BspEI20U/µl0,5
KpnI20U/µl0,5
Tango Buffer10x2
DNA300,4ng/µl4

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

pIG17_31 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-2
T4 Ligase -2
T4 Ligase Reaction buffer2x10
Transfer plasmid70ng/µl1
pIG17_03113ng/µl5

pIG17_34 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-2
T4 Ligase -2
T4 Ligase Reaction buffer2x10
Transfer plasmid70ng/µl1
pIG17_03415ng/µl5

pIG17_37 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-2
T4 Ligase -2
T4 Ligase Reaction buffer2x10
Transfer plasmid70ng/µl1
pIG17_03713ng/µl5

pIG17_87 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-2
T4 Ligase -2
T4 Ligase Reaction buffer2x10
Transfer plasmid70 ng/µl1
pIG17_0875ng/µl5

2.7.17

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

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
pIg17_0130,05pmol1
SV400,05pmol1
Neomycin0,05pmol1
pA0,05pmol1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
pIg17_0130,05pmol1
SV400,05pmol1
Zeocin0,05pmol1
pA0,05pmol1

03.07.17

Gibson assembly of SV40-TetR-pA

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol2
SV400,05pmol1
VP16-TetR0,05pmol1
pA0,05pmol1

7.7.17

Mini Prep of pIG17_031/034/086/119

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

concentration
pIG17.03146,6ng/μl
pIG17.03445,7ng/μl
pIG17.08647,9ng/μl
pIG17.119390,8ng/μl

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

pMF111 #1
Total rection volume 25µl

componentconcentrationvolume [µL]
H20-9
oIG17_177 (Primer fw)10µM1,25
oIG17_211 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

pMF111 #3
Total rection volume 25µl

componentconcentrationvolume [µL]
H20-9
oIG17_150 (Primer fw)10µM1,25
oIG17_176 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

pSB1C3 #2
Total rection volume 25µl

componentconcentrationvolume [µL]
H20-9
oIG17_198 (Primer fw)10µM1,25
oIG17_197 (Primer rv)10µM1,25
5x Q5 MM-12,5
DNA10ng/µl1

Gel picture

Midiprep of 31, 34, 86, 119

Midiprep with Promega Midiprep kit

pIG17_0316,0ng/μl
pIG17_0344,4ng/μl
pIG17_086 9,4ng/μl 6,9ng/μl
pIG17_119 156ng/μl 167ng/μl

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

componentconcentrationvolume [µL]
2x Gibson MM-6
pcDNA3.10,05pmol1
TetO0,05pmol1
HIF0,05pmol1
LUC0,05pmol1
pA0,05pmol1
pIG17_0580,05pmol1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
HRE0,05pmol1
HIF0,05pmol1
pA0,05pmol1
pIG17_0580,05pmol1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
CRE0,05pmol1
HIF0,05pmol1
pA0,05pmol1
pIG17_0580,05pmol1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
NFAT b.s. 3300,05pmol1
HIF0,05pmol1
pA0,05pmol1
pIG17_0580,05pmol1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
pcDNA3.10,05pmol1
NFAT b.s. 3800,05pmol1
HIF0,05pmol1
pA0,05pmol1
pIG17_0580,05pmol1

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_11851,6ng/μl
pIG17_120 #150ng/μl
pIG17_120 #256ng/μl
pIG17_121 #1208,3ng/μl
pIG17_121 #256ng/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 zeo107,9ng/μl
pIG17_031 neo25,7ng/μl
pIG17_097 1 #130,8ng/μl
pIG17_097 1 #234,2ng/μl
pIG17_097 2 #138,4ng/μl
pIG17_097 2 #240,0ng/μl
pIG17_097 2 #332,3ng/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 TetR22,0ng/μl
TetO-Luc-HIF #132,4ng/μl
TetO-HIF-Luc #1174,5ng/μl

13.7.17

Mini Prep of pCRE-HIF/CTLA4-HIF

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

concentration
pCRE-HIF #328,3ng/μl
pCRE-HIF #230,1ng/μl
pCRE-HIF #130,4ng/μl
CTLA4-HIF(380) #132,1ng/μl
CTLA4-HIF(380) #233,1ng/μl
CTLA4-HIF(380) #332,8ng/μl
CTLA4-HIF(330) #229,9ng/μl

15.7.17

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

HRE*4 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-6
T4 Ligase -2
T4 Ligase Reaction buffer2x10
pIG17_03165ng/µl1
HRE*410ng/µl1

CRE*4 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-6
T4 Ligase -2
T4 Ligase Reaction buffer2x10
pIG17_03165ng/µl1
CRE*410ng/µl1

NFAT bs*4 Total reaction volume 20µl

componentconcentrationvolume [µL]
H20-6
T4 Ligase -2
T4 Ligase Reaction buffer2x10
pIG17_03165ng/µl1
NFAT bs*410ng/µl1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
Transfer vector0,05pmol2
HRE*40,05pmol1
CFP0,05pmol1
pIG17_0580,05pmol1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
Transfer vector0,05pmol2
CRE*40,05pmol1
CFP0,05pmol1
pIG17_0580,05pmol1

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

componentconcentrationvolume [µL]
2x Gibson MM-5
Transfer vector0,05pmol2
NFAT bs*40,05pmol1
CFP0,05pmol1
pIG17_0580,05pmol1

19.7.17

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

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

concentration
pCRE-HIF26,7ng/μl
pCRE-HIF187,7ng/μl
pCRE-HIF153,0ng/μl
pCRE-HIF102,0ng/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_096342,8ng/μl
pIG17_09631,3ng/μl
pIG17_09671,3ng/μl
pIG17_09699,7ng/μl
pIG17_09655,8ng/μl

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

plasmid number primer
pCRE-HIFoIG17_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_096oIG17_174/175/169/170/018
pIG17_097oIG17_054
pIG17_118oIG17_019
pIG17_120oIG17_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

concentrationcurve
HRE x Mult-GFP-CMV-mCherry #139,4ng/μl
HRE x Mult-GFP-CMV-mCherry #228,9ng/μl
HRE x Mult-GFP-CMV-mCherry #328,9ng/μl
HRE x Mult-GFP-CMV-mCherry #444,6ng/μl
CRE x Mult-GFP-CMV-mCherry #118,9ng/μl
CRE x Mult-GFP-CMV-mCherry #249,8ng/μl
CRE x Mult-GFP-CMV-mCherry #320,9ng/μl
CRE x Mult-GFP-CMV-mCherry #425,2ng/μl
HRE ecFP zeo #135,2ng/μl
HRE ecFP zeo #226,4ng/μl
HRE ecFP zeo #336,2ng/μl
HRE ecFP zeo #417,3ng/μl
TetR VP16 neo #121,7ng/μl
TetR VP16 neo #230,4ng/μl
TetR VP16 neo #366,7ng/μl
TetR VP16 neo #428,1ng/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

concentrationcurve
pIG17_012187,1ng/μl
pIG17_01353,9ng/μl
pIG17_01549,1ng/μl
pIG17_01626,1ng/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_00316,9ng/μl
pIG17_00390,8ng/μl
pIG17_003144,0ng/μl
pIG17_004154,9ng/μl
pIG17_004280,7ng/μl
pIG17_031541,1ng/μl (!)
pIG17_031257,1ng/μl (!)
pIG17_031336,1ng/μl (!)
pIG17_03423,0ng/μl (!)
pIG17_119318,6ng/μl (!)
pIG17_11919,3ng/μl
pIG17_11931,9ng/μl
pIG17_11912,1ng/μl
pIG17_11953,2ng/μl
pIG17_119314,0ng/μl (!)
HRE GFP109,9ng/μl
HRE GFP43,9ng/μl
HRE GFP89,9ng/μl
HRE GFP147,1ng/μl
HRE GFP104,3ng/μl
HRE GFP67,9ng/μl (!)
HRE GFP125,2ng/μl
HRE GFP153,1ng/μl
HRE GFP318,2ng/μl
HRE GFP294,9ng/μl
HRE GFP148,6ng/μl
HRE GFP82,1ng/μl
HRE GFP423,9ng/μl (!)
HRE GFP427,2ng/μl (!)

30.7.17

Mini Prep of pIG17_503/Tet-GFP

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

concentrationcurve
pIG17_503242,6ng/μl
Tet-GFP255,1ng/μl