Team:Freiburg/Notebook/lab multiple targeting

from 6 colonies with pIG7001a (Gibson Assembly 28-05)

Program

30 cycles

• Temp.: 37°C
• Incubation time: 1h

PCR 1

colonie 1 was used (oIG7001a)
for Gibson Assembly 2:

5µl of each product were load on a gel

Products of PCR 1

Product 3 from Gibson 2 is missing → 0,2 µl extra plasmid template was added for PCR 2

PCR 2

for gibson assembly 2, 3, and 4
template: products from 1. PCR

concetration of PCR products:
Gibson 2: 1415 ng/µl, 1417 ng/µl, 1315 ng/µl
Gibson 3: 2874 ng/µl, 1436 ng/µl, 337 ng/µl
Gibson 4: 324 ng/µl, 325 ng/µl, 2047 ng/µl

5µl of each PCR product were load on a gel

Products of PCR 2

Gibson Assembly

volumes were calculated with excel sheet with concentration and length:
used volumes:
Gibson 2: 0,3 µl, 0,3 µl, 0,1 µl
Gibson 3: 0,15 µl, 0,3 µl, 0,4 µl
Gibson 4: 1,4 µl, 1,4 µl, 0,1 µl
1h in 1 aliquot of Gibson mix (according to protocol)
→ pIG7002a, heat shock transfection in E. coli (according to protocol)
→ pIG7003a, heat shock transfection in E. coli (according to protocol)
→ pIG7004a, heat shock transfection in E. coli (according to protocol)

31.05.13

Digest of pIG7001a

used: colonie 1 (pIG7001a), NcoI, PstI HF, XbaI

expected bands

pIG7001a digest left to right: 1: ladder (1kb) 2: XbaI 3: NcoI 4: PstI 5: negative control

XbaI did only cut once instead of twice, NcoI and PstI cut as expected

Sequencing of pIG7001a(1)

GATC-Watch-box: 706894
→ Sequence of pIG7001a ok

June

01.06.13

Repetition of gibson assembly 2,3,4 with old PCR products new 2. PCR only for fragment 3 of gibson 3 –> contamination with plasmid template

PCR 2 (fragment 3 / gibson 3)

same protocol was used 5µl of PCR product were load on gel

Products of PCR 2 Fragment 3, Gibson 3

Gibson Assembly

volumes were calculated with excel sheet only by length:
used volumes:
Gibson 2: 1,5 µl, 1,5 µl, 0,5 µl
Gibson 3: 1,5 µl, 1,5 µl, 0,5 µl
Gibson 4: 1,5 µl, 1,5 µl, 1,0 µl

→ pIG7002a, heat shock transfection in E. coli (according to protocol)
→ pIG7003a, heat shock transfection in E. coli (according to protocol)
→ pIG7004a, heat shock transfection in E. coli (according to protocol)

02.06.13

6 colonys each (pIG7002a,pIG7003a, pIG7004a) were picked and plated
2 colonys control were picked and plated

03.06.13

Doubledigest of pIG7002a, pIG7003a, pIG7004a

PstI and HindIII

Expected bands

Digest of pIG7002a, pIG7003a, pIG7004a with PstI and HindIII

• For pIG7002a only the plasmid form colony 4 shows expected bands.
• No colony from pIG7003a is positive.
• Only the plasmid from colony 4 of pIG7004a shows the expected bands.

Sequencing of pIG7002a (4) and pIG7004a (4)

GATC-Watch-box: 707852
→ Sequence of pIG7002a ok
→ Frameshift in pIG7004a ok

04.06.13

6 Minipreps of 6 new colonys with pIG7003a: (7)-(12)

Doubledigest of pIG7003a

PstI and HindIII

Digest of pIG7003a (7)-(12)

05.06.13

Sequencing of pIG7003a (12)

→ Sequence of pIG7003a (12) not ok: mito-sequence missing

06.06.13

6 colonys od pIG7004a were picked and plated

07.06.13

6 Minipreps of 6 new colonies of pIG7004a (7)-(12)

Doubledigest of pIG7004a

PstI and HindIII

Sequencing of pIG7004a (7)

→ Sequence of pIG7004a (7) ok

11.06.13

12 colonys od pIG7003a were picked and plated

12.06.13

12 Minipreps of 12 new colonys with pIG7003a(13) - (24)

Doubledigest of pIG7003a

BamHI and AflII

Digest of pIG7003a (13)-(24) with BamHI and AflII

Sequencing results of pIG7001(1), pIG7002(4), pIG7004(7)

→ tetO13 sequence is not complete in pIG7001(1), pIG7002(4) and pIG7004(7)

Sequencing of pIG7003a(13) and (14)

→Sequence of pIG7003a (13) not ok: promotor and mito sequences missing, tetO13 not complete
→Sequence of pIG7003a (14) not ok: mko missing, tetO 13 not complete

13.06.13

Repetition of Gibson assembly 3 with new pcr products

PCR 1

Fragment 1	pIG7001a	oIG7004-F	oIG7004-R	2274 bp
Fragment 2	pIG7001a	oIG7005-F	oIG7005-R	2248 bp
Fragment 3	pFucci-G1-Orange	oIG7007-F	oIG7007-R	659 bp

PCR program

98°C	5min	Denaturation
98°C	30s	Denaturation
60°C	30s	Annealing
72°C	40s/kb	Elongation
72°C	10min	final Elongation
4°C	hold

15 cycles

no products visible --> PCR did not work

14.06.13

Repetition of Gibson assembly 3 with new pcr products

PCR 1

Fragment 1	pIG7001a	oIG7004-F	oIG7004-R	2274 bp
Fragment 2	pIG7001a	oIG7005-F	oIG7005-R	2248 bp
Fragment 3	pFucci-G1-Orange	oIG7007-F	oIG7007-R	659 bp

PCR program

98°C	5min	Denaturation
98°C	30s	Denaturation
60°C	30s	Annealing
72°C	35s/kb	Elongation
72°C	10min	final Elongation
4°C	hold

20 cycles

PCR 2

template: products of PCR 1

PCR program

98°C	5min	Denaturation
98°C	30s	Denaturation
60°C	30s	Annealing
72°C	35s/kb	Elongation
72°C	10min	final Elongation
4°C	hold

20 cycles

all products visible

Gibson Assembly

volumes were calculated with excel sheet with concentration and length:
used volumes:
Gibson: 0,88µl, 0,88µl, 0,2µl 1h in 1 aliquot of Gibson mix (according to protocol)
→ pIG7003a, heat shock transfection in E. coli (according to protocol)

15.06.13

3 colonys of pIG7003a were picked and plated

17.06.13

3 Minipreps of 3 colonys (pIG7003a(25)-(27))

Doubledigest of pIG7003a

BamHI and AflII

Digest of pIG7003a (25)-(27) with BamHI and AflII

Restrictiondigest of pIG7001a, pIG7002a, pIG7004a, pKM006

EcoRV and NruI

Restrictiondigest with EcoRV and NruI left to right: 1:pIG7001a 2:pIG7002a 3:pIG7004a 4:pKM006

• Bands are to weak for quadruple ligation with pKM006

Gibson Assembly

Repetition of the Gibson Assembly from 14.06.2013
used volumes (products from PCR2):
Gibson: 0,88µl, 0,88µl, 0,2µl 1h in 1 aliquot of Gibson mix (according to protocol)
→ pIG7003a, heat shock transfection in E. coli (according to protocol)

18.06.13

Gibson Assembly

no colonies on Gibson plate from 17.06.2013 → Repetition of the Gibson Assembly from 14.06.2013
used volumes (products from PCR2):
Gibson: 0,9µl, 0,9µl, 0,4µl 1h in 1 aliquot of Gibson mix (according to protocol)
→ pIG7003a, heat shock transfection in E. coli (according to protocol)

Restrictiondigest of pIG7001a, pIG7002a, pIG7004a, pKM006

Repetition of the restrictiondigest from 17.06.2013
EcoRV and NruI

Restrictiondigest with EcoRV and NruI left to right: 1:pIG7001a 2:pIG7002a 3:pIG7004a 4:pKM006

• Bands for the backbones (big fragments) are cut out of pIG7001a, pIG7002a and pIG7004a lane
  and tet013 (small fragment) is cut out of the pKM006 lanes
• → Gelextraction of gel slices (according to protocoll)
  concentrations:
  pIG7001a= 13 ng/µl
  pIG7002a= 10 ng/µl
  pIG7004a= 14 ng/µl
  pKM006= 1,3-1,5 ng/µl

Ligation of pIG7001a, pIG7002a and pIG7004a

Insert	pKM006	11µl
Backbone	pIG7001a; pIG7002a; pIG7004a	11µl
10x Puffer	2,5µl
T4 DNA Ligase	1µl

• Incubation for 15 minutes (RT)

→ pIG7001b, pIG7002b, pIG7004b, heat shock transfection in E. coli (according to protocol)
used: 5µl Ligationmix for 50µl TOP10 cells

19.06.13

5 colonies picked and plated for pIG7003a (Gibson)
6 colonies each picked and plated for pIG7001b, pIG7002b, pIG7004b(Ligation)

Seeding of HELAs

for transfection

• count: 22*10^4 cells/ml
• used volume (24 well plate): 0,5 ml/well
• used concentration (24 well plate): 8*10^4 cells/well → 8*10^4[cells/ml] / 0,5[ml] =16*10^4 [cells/ml]
• 12 wells needed → 6ml total needed

calculation:
c1*v1=c2*v2
v1=(c2*v2)/c1
v1=(16*10^4 [cells/ml] * 6 [ml])/ 22*10^4 [cells/ml]
v1=4,3 ml
→ 4,3 ml cells + 1,7 ml medium

Inoculation of liquid cultures

for MIDIprep with pIG7001a, pIG7002a, pIG7004a
used: 100ml LB-medium,100µl kanamycin

20.06.13

Miniprep of 5 colonies with pIG7003a(28-32)
Miniprep of 6 colonies each with pIG7001b(1-6), pIG7002b(1-6), pIG7004b(1-6)
Midiprep of pIG7001a, pIG7002a, pIG7004a → no template

Doubledigest of pIG7003a

BamHI and AflII

Doubledigest with BamHI and AflII

Doubledigest of pIG7001b, pIG7002b, pIG7004b

EcoRV and PstI

Doubledigest with EcoRV and Pst

Sequencing of pIG7003a (29) and (30)

→ Sequence not ok, mito sequence missing

Transfection of HELAs

with pIG7001a, pIG7002a and pIG7004a + pSAM200 (according to protocol)
→ no fluorescence visable

21.06.13

Doubledigest of pIG7001b, pIG7002b, pIG7004b

EcoRI and PstI

Doubledigest with EcoRI and Pst

22.06.13

Repetition of Gibson Assembly for pIG7003a with new strategy

PCR

Fragment 1	pIG7001a	oIG7004-F	oIG7004-R	2274 bp
Fragment 2	pIG7001a	oIG7005-F	oIG7005-R	2248 bp
Fragment 3	pFucci-G1-Orange	oIG7007-F	oIG7007-R	659 bp

PCR program

98°C	5min	Denaturation
98°C	30s	Denaturation
60°C	30s	Annealing
72°C	35s/kb	Elongation
72°C	10min	final Elongation
4°C	hold

15 cycles

Gelextraction

23.06.13

4 colonies are picked and plated

24.06.13

Miniprep of pIG7003a(1-4)

Doubledigest of pIG7003a (1-4)

EcoRV and NruI

Restrictiondigest with EcoRV and NruI left to right 1:Marker 2,3,4,5:pIG7003a(1-4)

Sequencing of pIG7003a(4)

→ Sequence not ok, mito Sequence missing

27.06.13

Gelextraction

Restrictiondigest with EcoRV and NruI left to right 1:Marker 2,3:pIG7002a 4,5:pIG7004a

Bands for the backbones are cut out

Restrictiondigest with EcoRV and NruI left to right 1:Marker 2,3:pKM006

Bands for tetO13 are cut out

July

13.07.13

Repetition of the restrictiondigest (pIG7001a, pIG7002a, pIG7004a and pKM006)
EcoRV and NruI
Bands for the backbones (big fragments) are cut out of pIG7001a, pIG7002a and pIG7004a lane and tet013 (small fragment) is cut out of the pKM006 lanes

15.07.13

Ligation of pIG7001b, pIG7002b and pIG7004b

Insert	pKM006	11µl
Backbone	pIG7001a; pIG7002a; pIG7004a	11µl
10xBuffer		2,5µl
T4 DNA Ligase		1µl

• incubation for 15 minutes (RT)

→ pIG7001b, pIG7002b, pIG7004b, heat shock transfection in E. coli (according to protocol)
used: 5µl Ligationmix for 50µl TOP10 cells

16.07.13

-2 colonies picked and plated for pIG7001b, pIG7002b and pIG7004b in liquid culture
Miniprep of pIG7001b (1-2), pIG7002b (1-2), pIG7004b (1-2)

18.07.13

Doubledigest of pIG7001b(1-2), pIG7002b(1-2), pIG7004b(1-2)

EcoRV and NheI

Restrictiondigest with EcoRV and NheI left to right 1:pIG7001b(1) 2:pIG7001b(2) 3:pIG7002b(1) 4:pIG7002b(2) 5:pIG7004b(1) 6:pIG7004b(2)

Sequencing of pIG7001b(2), pIG7002b(2), pIG7004b(2)

→ Sequence ok

19.07.13

Changing of the target cassett in two constructs (pIG7002b and pIG7004b)
→ every construct can be targeted seperately

PCR

amplifying the 400bp cassett out of the opposite construct (pIG7002 insert for pIG7004b backbone; pIG7004 insert for pIG7002b backbone)

Fragment 1	pIG7002b	oIG7009-F	oIG7009-R	400bp
Fragment 2	pIG7004b	oIG7011-F	oIG7011-R	400bp

PCR program

98°C	5min	Denaturation
95°C	30s	Denaturation
60°C	30s	Annealing
72°C	30s/kb	Elongation
72°C	10min	final Elongation
4°C	hold

15 cycles

→ PCR product 2 and 4

22.07.13

Restrictiondigest of pIG7002b, pIG7004b, PCR product 2 and PCR product 4

DNA	Enzyme
pIG7002b	NruI and NheI
pIG7004b	PstI and NheI
PCR product 2	PstI and NheI
PCR product 4	NheI

Restricted DNA was load on gel:

Restrictiondigest with EcoRV and NheI left to right 1:pIG7002b 2:PCR product 2 3:pIG7004b(1) 4:PCR product 4

→ no bands for both PCR products visible, pIG7002b and pIG7004b: restricted DNA band not differentiable from not restricted

23.07.13

- Repetition of PCR (19.07.) and Restriction (22.07.)

Fragment 1	pIG7002b	oIG7009-F	oIG7009-R	400bp
Fragment 2	pIG7004b	oIG7011-F	oIG7011-R	400bp

PCR program

95°C	30s	Denaturation
95°C	30s	Denaturation
60°C	30s	Annealing
68°C	30s/kb	Elongation
68°C	5min	final Elongation
4°C	hold

30 cycles

→ PCR product 2 and 4

24.07.13

Doubledigest of PCR product 2 and 4
- bands for both PCR products visible

Restrictiondigest of pIG7002b, pIG7004b, PCR product 2 and PCR product 4

DNA	Enzyme
pIG7002b	NruI and NheI
pIG7004b	PstI and NheI
PCR product 2	PstI and NheI
PCR product 4	NheI

Restricted DNA was load on gel:

Restrictiondigest left to right 1:Marker 2:PCR product 2 3:PCR product 4

Restrictiondigest left to right 1:Marker 2:pIG7002b 3:pIG7004b

Bands are cut out
Gelextraction concentrations:
pIG7002b: 10,7 ng/µl
pIG7004b: 31 ng/µl
PCR product 2: 14,4 ng/µl
PCR product 4: 3,3 ng/µl

Ligation

for pIG7002c

Stuff	Amount
restricted pIG7002b	5µl
restricted PCR product 4	4µl
T4 Ligase Buffer (10X)	2µl
T4 Ligase	1µl
water	8µl
total	20µl

for pIG7004c

Stuff	Amount
restricted pIG7004b	1,8µl
restricted PCR product 2	1µl
T4 Ligase Buffer (10X)	2µl
T4 Ligase	1µl
water	14,2µl
total	20µl

incubation for 30 minutes (RT)

→ pIG7002c, pIG7004c, heat shock transfection in E. coli (according to protocol)
used: 1µl Ligationmix for 25µl TOP10 cells

25.07.13

6 colonies each are picked and plated

26.07.13

Miniprep of all 12 colonies
Sequencing of the first 3 colonies each

27.07.13

Sequences not ok
Sequencing of the last 3 colonies each

28.07.13

Sequences not ok
→ Ligation failed

August

12.08.13

Digest of reporter plasmids

µl	type
3 µg	DNA (pKM602, pKM608, pKM611)
5	NEB-Buffer 4
1	Nhe I HF
1	Ssp I HF
0,5	BSA
Add to 50µl	H2O

• Temp.: 37°C
• Incubation time: 2h

µl	type
3 µg	DNA (pIG7001b, pIG7002b, pIG7004b)
5	Promega MC Buffer
1	Promega Nhe I
2	Promega Nru I
0.5	BSA
Add to 50µl	H2O

• Temp.: 37°C
• Incubation time: o/n

13.08.13

Gel run

A) Digest of pIG700..

From left to right: Marker (1 kb Roth); pIG7001; pIG7002; pIG7004 (50 & 4 µl); pIG7004 undigested (4 µl)

From left to right: Marker (1 kb Roth); pIG7001; pIG7002; pIG7004 (50 & 4 µl); pIG7004 undigested (4 µl)

B) Digest of pKM60..

From left to right: Marker (2 log NEB); pKM602; pKM608; pKM611 (50 & 4 µl)

From left to right: Marker (2 log NEB); pKM602; pKM608; pKM611 (50 & 4 µl)

All bands are at the expected sizes.

Gel extraction

DNA were purified using High Pure Plasmid Isolation Kit of Roche.

Changes to the protocol:

• incubation at 56 °C for 10 min for gel dissolving
• elution with dH₂O (incubation at 50 °C for 4 min before centrifugation)

Yield: 10 ng/µl (pIG700..); 2 ng/µl (pKM60..)

Recombination of cutted parts

ingredient	amount
pIG7001/2	2.5 µl
pKM602/11	2 µl
T4-Ligase	1 µl
T4-Ligase buffer	2 µl
dH2O	up to 20 µl

Incubation at 22 °C for 1 h.

ingredient	amount
pIG7004	4.3 µl
pKM608	1.2 µl
T4-Ligase	1 µl
T4-Ligase buffer	2 µl
dH2O	up to 20 µl

Incubation at 22 °C for 1 h.

Trafo

1. 4 µl of plasmid were added to 25 µl of chemically competent E. coli cells
2. incubation for 10 min on ice
3. heatshock (42 °C for 45 s)
4. incubation for 2 min on ice
5. addition of 300 µl LB medium
6. incubation for 1 h at 37 °C (shaking)
7. distribution of 300 µl on LB plates with kanamycin
8. incubation over night at 37 °C

14.08.13

Picking of clones

From each Ligation 7 clones were picked and spread on 1/4 plates.

15.08.13

Miniprep

DNA was purified using High Pure Plasmid Isolation Kit of Roche.

Yield: ~ 220 ng/µl

Test digest

ingredient	volume
plasmids (220 ng/µl)	1.2 µl
EcoRV-HF	0.5 µl
NEB buffer 4	1 µl
dH2O	up to 10 µl

Incabation at 37 °C for 2 h.

Gel run

From left to right: Marker (1 kb Roth); pIG7005 (3x); pIG7006 (3x); pIG7007 (3x)

pIG7005_2, all clones of pIG7006 and pIG7007_1 & 3 showed the expected bands. pIG7005_2, pIG7006_1 and pIG7007_3 were send in for sequencing.

16.08.13

Midiprep

Though the sequencing did not have a result, plasmids were amplified and midiprepped by Pure Yield Plasmid Midiprep System from Promega because of the results of the test digest.

Seeding of cells for microscopy

A 24 well plate with cover slips was filled with 50,000 cells per well.

17.08.13

Transfection

Protocol:

1. 40 µl Opti-MEM + 1.5 µl PEI-solution were mixed in a 1.5 ml Eppi.
2. 0.5 µg of the DNA of interest were prepaired in another Eppi .
3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
4. Solution was spread drop-wise to the cells in the dish

Transfection scheme:

18.08.13

Fixation

Cells were washed with 500 µl PBS per well and fixated with 200 µl PFA per well (incubation on ice for 45 min).

Cover slips were washed in dH₂O and mounted on a drop of Mowiol with DABCO on a object slide.

19.08.13

Flourescence microscopy

GFP and mCherry were expressed in all cells, only in the cells with TetR-VP16 there was a stronger signal. BFP was not detectable because of the microscope.

21.08.13

Seeding of cells for microscopy

A 24 well plate with cover slips was filled with 50,000 CHO cells per well.

22.08.13

Transfection

Protocol:

1. 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
2. 0.75 µg of the DNA of interest were prepaired in another Eppi .
3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
4. Solution was spread drop-wise to the cells in the dish

Medium change

Medium was changed after 3 h.

23.08.13

Fixation

Cells were washed with 500 µl PBS per well and fixated with 200 µl PFA per well (incubation on ice for 45 min).

Cover slips were washed in dH₂O and mounted on a drop of Mowiol with DABCO on a object slide.

25.08.13

Flourescence microscopy

GFP and mCherry were detectable, but no differences in fluorescence intensity.

31.08.13

Seeding of cells for flow cytometry

A 24 well plate was filled with 50,000 HEK cells per well.

September

01.09.13

Transfection

Protocol:

1. 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
2. 0.75 µg of the DNA of interest were prepaired in another Eppi .
3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
4. Solution was spread drop-wise to the cells in the dish

DNA amount of effectors was 6 times higher than DNA amount of fluorescence proteins.

03.09.13

Preparation for flow cytometry

• Removal of medium.
• Washing with PBS.
• Detaching of the cells with 25 µl trypsin per well.
• Addition of 250 µl FACS buffer (PBS with 1 % FCS).
• Samples were filled in little FACS tubes.

Flow cytometry

Intensities of all flourescent protein were measured.

Retrieved from "http://2013.igem.org/Team:Freiburg/Notebook/lab_multiple_targeting"

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