Team:Freiburg/Notebook/lab multiple targeting

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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)

12.08.13

Digest of reporter plasmids

Product

pKM006

oIG7003-F

oIG7003-R

1100bp

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.

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.