Team:Freiburg/Notebook/lab multiple targeting

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Revision as of 15:12, 16 September 2013

crRNA - Multiple targeting

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

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.

18.08.13

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

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 + 1.5 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.

@@ Line 46: / Line 46: @@
 <p class="second_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_multiple_targeting"> Multiple targeting </a> </p>
+<p class="second_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_gfp_reporter"> GFP-reporter-plasmid </a> </p>
 <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/method"> uniBAss </a></p>