Team:Freiburg/Notebook/lab multiple targeting

From 2013.igem.org

Revision as of 14:17, 24 September 2013 by AnneM (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

crRNA - Multiple targeting

May

28.05.13

PCR 1

for Gibson Assembly

Fragment 1	pACGFP1-Golgi	oIG7001-F	oIG7001-R	2209 bp
Fragment 2	pACGFP1-Golgi	oIG7002-F	oIG7002-R	2126 bp
Fragment 3	pKM006	oIG7003-F	oIG7003-R	1100 bp

Stuff	Volume
Template(Plasmid)	0,5µl
Primer(10 µM)	each 1µl
Q5 Polymerase	0,5µl
dNTPs(2,5mM)	2µl
Q5 Buffer(5x)	4µl
Water	11µl
total	20µl

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

14 cycles

15µl of each product from PCR 1 were load on gel:

Products PCR 1
left to right:
1: ladder (1kb)
2: Fragment 1 (2209bp)
3:Fragment 2 (2126bp)
4:Fragment 3 (1100bp)

PCR 2

for Gibson Assembly
template: products from PCR 1

Stuff	Volume
Template(PCR product)	2µl
Primer(10 µM)	each 1µl
Q5 Polymerase	1µl
dNTPs(2,5mM)	5µl
Q5 Buffer(5x)	10µl
Water	30µl
total	50µl

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

18 cycles

Concentration of PCR-Products:
1: 990 ng/µl 2: 900 ng/µl 3: 870 ng/µl

4µl of each product from PCR 2 were load on gel:

Products PCR 2
left to right:
2: ladder (1kb)
3: Fragment 1 (2209bp))
4: Fragment 2 (2126bp)
5: Fragment 3 (1100bp)

Gibson Assembly

Calculation of DNA mix for Gibson Assembly:
concentration [ng/µl] * size [kbp] * 0.00023 [1/(ng*kb)]
1h in 1 aliquot of Gibson mix (according to protocol)
→ pIG7001a , heat shock transfection in E. coli (according to protocol)

29.05.13

6 colonys pIG7001a were picked and plated

Colony PCR

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

Product

pKM006

oIG7003-F

oIG7003-R

1100bp

Volume	Stuff
	Template (Bacteria from colonie)
1µl	Taq Standart buffer (10x)
0,5µl	Primer1
0,5µl	Primer2
2.5µl	dNTPs
0.5µl	Taq polymerase
7µl	H₂O
10µl	total

Program

98°C	7min	Denaturation
98°C	30s	Denaturation
67°C	30s	Annealing
72°C	50s	Elongation
72°C	10min	final Elongation
4°C	hold

30 cycles

10µl of each PCR product were load on a gel:

Products Colony-PCR
left to right:
1: ladder (1kb)
2-7: colonie 1-6 from pIG7001a (gibson assembly 05-28)
8: negativ control
9: positive control (template is pKM006)

PCR did not work

30.05.13

6 minipreps of pIG7001a:
cancentrations: 196 ng/µl, 175 ng/µl, 195 ng/µl, 133 ng/µl, 203 ng/µl, 197 ng/µl

Double-Digest of pIG7001a

used: minipreps of colonies 1-6, NgoMIV, PstI HF

µl	type
5	DNA
1	NEB-Buffer 4
0.5	Pst1 HF
0.5	NgoMIV
Add to 10 µl	H₂O

Temp.: 37°C
Incubation time: 1h

the whole mix was load on a gel:

expected bands

Products double digest:
pIG7001a (Prep 1-6) with Pst1 and NgoMIV

PCR 1

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

Fragment 1	pIG7001a	oIG7004-F	oIG7004-R	2274 bp
Fragment 2	pIG7001a	oIG7005-F	oIG7005-R	2248 bp
Fragment 3	GW1-Peredox_mCherry-NLS	oIG7006-F	oIG7006-R	741 bp

for Gibson Assembly 3:

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

for Gibson Assembly 4:

Fragment 1	pIG7001a	oIG7004-F	oIG7004-R	2274 bp
Fragment 2	pIG7001a	oIG7005-F	oIG7005-R	2248 bp
Fragment 3	pDS47-BFP-dGEM-trunc	oIG7008-F	oIG7008-R	679 bp

µl	type
4	Q5-HF Reaction Buffer
0,5	Template
1	each Primer
5	dNTPs
0.5	Q5-HF Polymerase
Add to 20	H₂O

Gibson 1 program was used

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

µl	type
4	Q5-HF Reaction Buffer
2	Template
1	each Primer
5	dNTPs
0.5	Q5-HF Polymerase
Add to 20	H₂O

Gibson 2 program was used

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

µl	type
5	DNA
1	NEB-Buffer 4
0,5	XbaI
0,1	BSA
Add to 10µl	H₂O

µl	type
5	DNA
1	NEB-Buffer 4
0,5	NcoI HF
Add to 10µl	H₂O

µl	type
5	DNA
1	NEB-Buffer 4
0,5	PstI HF
Add to 10µl	H₂O

Temp.: 37°C
Incubation time: 1h

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

div id="tag">

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

div id="tag">

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

div id="tag">

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)

div id="tag">

15.06.13

3 colonys of pIG7003a were picked and plated

div id="tag">

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)

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)

B) Digest of pKM60..

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

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

40 µl Opti-MEM + 1.5 µl PEI-solution were mixed in a 1.5 ml Eppi.
0.5 µg of the DNA of interest were prepaired in another Eppi .
Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
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:

40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
0.75 µg of the DNA of interest were prepaired in another Eppi .
Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
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:

40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
0.75 µg of the DNA of interest were prepaired in another Eppi .
Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
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.