Team:Freiburg/Notebook/method

From 2013.igem.org

(Difference between revisions)

Revision as of 19:21, 28 September 2013

uniBAss - uniCAS Binding Assay Notebooks

Labbook Development of the uniBAss

May

Digest with BbsI

µl	type
8	pX334a (243ng/µl)
2µl	NEB-Buffer 2.1
1µl	BbsI
Add to 50 µl	H₂O

pX334a was digested for 2h at 37°C using BbsI for subsequent target insertion

Gel extraction of the digested pX334a

Bands were cut out and extracted with Roche high pure PCR product purification kit. Eluation in 20 µl H₂O, before centrifugation: incubated 10min at room temp. Eluted liquid put on column again, incubated for 10 min and centrifuged. concentrations: less than 16.5 ng/ul.

Ligation of digested pX334a with annealed oIG2007-oIG2008 (EMX1 locus)

µl	type
2.5	pX334a (16.5 ng/µl)
1µl	T4 ligase buffer
0.5µl	T4 ligase

Five different ligation mixes (dilution row of insert, 1x, 10x 100x ,500x, 1000x diluted insert) + the negative control with water instead of oligo were incubated for 1h at 22°C. This resulted in pIG9000.

Transformation of Top10 E.coli with the pX334a containing the EMX1 locus (pIG9000)

The 6 different samples were transformed following the protocol and were thus given on an agar plate with Ampicilline and incubated over night

16. May

Picking colonies and streakout

The negative control showed five colonies whereas the plate with 1x duluted insert had 12 colonies, 10x had 17 colonies, 100x had 17 colonies, 500 had 10 colonies, 1000x hat 18 colonies. From each of the two plates with the most colonies were 8 colonies picked. 16ten streakouts were performed on AMP agar plates and incubated over night at 37°C

17. May

Miniprep and testdigest

The miniprep was performed as stated in the protocol. Afterwards a testdigest was performed

Testdigest of 16. possible pIG9000 colonies, mastermix contained

µl	type
total 40µl	Sample
4µl	BbsI
4µl	NcoI
20µl	NEB 2.1
132µl	H2O

after 2h at 37°C the samples were applied on an agarose gel (1% Agar, 0.5 TAE to 50 ml) to visualize band 3 ul gel red was used. The gel displayed that some colonies showed no additional band and therefore are likely to have the insert included. Those positive minipreps were send to sequencing at GATC

The sequencing results validated that the insert was successfully cloned into pX334a and therefore pIG9000 was ready for the testing on the ELISA.

18. May

Oligoannealing and transformation of E.coli with pIG9000 with the aim the perform a midi prep

The oligoannealing with the biotinylated oligonucleotides oIG9000, oIG9001 and oIG9002 was performed at 95°C for 5 minutes, afterwards the heating block was turned off. The transformation was performed following the protocol. The transformed cells were given into 150ml LB-media containing AMP and were incubated overnight at 37°C

19. May

Performing a midiprep

A midiprep of the E.coli containing pIG9000 was performed following the protocol. Result: 2487ng/ul in a volume of 200ul.

20. May

Seeding cells for transfection

HEK293T cells were seeded at a density of 250.000 cells/ml. The conservation culture contained 60.000 cells/ml.

Human embryonic kidney cells (HEK-293T) were cultivated in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10 % Fetal Calf Serum (FCS) and 1 % penicillin / streptomycin solution (DMEM complete) and incubated in a moisturized atmosphere containing 5 % CO2. Cells were detached with 2.4 ml Trypsin-EDTA solution, transferred into 7 ml fresh medium and centrifuged for 3 min at 900 x g. The supernatant was removed and the pelleted cells were resuspended in 5 - 7 ml medium. Cell numbers were determined by transferring 100 μl cell solution to 10 ml of Casyton Buffer and counted in an Innovatis Casy Cell counter (Casy-Technology, Reutlingen, Germany, model TT). For maintaining cultures, HEK-293T cells were cultivated in petridishes with a starting concentration of 0.6 or 1.2*106 cells in 10 ml medium and incubated as described before.

21. May

Contamination & seeding cells

Contamination occured therefore the cells had to be seeded again following the protocol.

23. May

PEI Transfection and preparation of uniBAss experiment

Seeded cells were transfected with pIG9000 following the protocol Cells were transfected using the PEI transfection method with the following concentrations: Typically for a 200 µl transfection mix, 1.5 µg of DNA and 5 µl PEI (Polysciences, Inc., PA, cat. no. 23966) were filled up with OptiMEM (Invitrogen GmbH, Darmstadt, Germany cat. no. 11058-021) to 100 µl. Afterwards, the 100 µl PEI solution was added to the 100 µl DNA solution, vortexed and incubated at RT for 15 to 30 min. Afterwards 200 µl of the PEI / DNA transfection mix was added drop-wise to each 1 ml culture medium containing cells in well plates and incubated at 37 °C, 5 % CO2. After 5 h, the medium was exchanged , Dulbecco's modified Eagle's medium (DMEM), supplemented with 10 % Fetal Calf Serum (FCS) and 1 % penicillin / streptomycin solution (DMEM complete)

The ELISA plates (Corning, Inc., cat. no. 3590, NY, New York) were coated with 100 µl streptavidin solution (20 µl / 10 ml in dH20 to obtain a final concentration of 4 µg / ml) and incubated overnight at 4°C.

24. May

uniBAss experiment to obeserve the influence of different Ion concentrations

The streptavidin coated plates were washed 3 x with TBST (TBS, 2 ml / l Tween20) and 300 µl blocking buffer (1 x TBST, 1 % BSA) was added for 1 h. After 3 x washing with TBST, the biotinylated oligonucleotides (10pmol/well) were applied and incubated at RT for 1 h. Afterwards the plates were washed 3 x with TBST. To obtain the cell lysate with the Cas9 protein, the transfected HEK-293T cells (250.000 cells / ml) were resuspended in 250 µl dilution buffer (10 mM Tris, 1 % BSA, 10 mM MgCl2, 10 mM NaCl) containing EDTA free Protease inhibitor per 125.000 cells / ml and sonified for 10 min. To remove the cell fractions the lysate was centrifuged for 5 min at 500 g. To adjust the buffer condition a dilution row with cell lysate comprising the Cas9 were performed in round 96-well plates. The dilution buffers used were b1 (10 mM Tris, 1 % BSA, 10 mM MgCl2, 250 mM NaCl) and b2 (10 mM Tris, 1 % BSA, 100 mM MgCl2, 10 mM NaCl) respectivly. This resulted in the following conditions.

MgCl2 [mM]	NaCl [mM]
10	250
10	125
10	62.5
10	31.25
10	15.62
10	7.81
10	3.9
10	1.95
10	0.97
10	0.48

NaCl [mM]	MgCl2 [mM]
10	100
10	50
10	25
10	12.5
10	6.25
10	3.12
10	1.56
10	0.89
10	0.39
10	0.19

Afterwards 100 µl of the diluted cell lysate was transferred to the ELISA plates. After 1 h incubation at RT the plates were washed 3 x with TBST and 100 µl anti-HA antibody solution (1000 x diluted in blocking buffer) was applied to each wells and incubated for 1 h. After 3 x washing with TBST, 100 µl anti-mouse HRP antibody (4000 x diluted in blocking buffer,) were added. After 1 h incubation, plates were washed 3 x with TBST, probed with 100 µl ELISA ABTS substrate and the absorbance was measured at 405 nm. For the positive controls, wells were coated with 100 µl FM protein (1.0 µg / ml) and directly with the sonificated cell lysate in the dilution buffer. The negative controls performed were composed of, no Antibody, no oligonucleotide at all, not the matching oligonucleotide sequence.

in the following this protocol was always used only variations are mentioned

Result: XXX Figure1

Binding restrain of the Cas9 MX1 crRNA complex for bisected buffer

To check if the Cas9 protein was in the cell lysate a westernplot was performed

For specific detection of proteins, semi-dry Western Blots were performed. The polyvinylidene fluoride membrane (PVDF) (0.45 µm, Millipore Corporation, Bedford, MA, cat. no. T831.1) was first activated in methanol then washed with dH2O and subsequently equilibrated in the transfer buffer. For Semi-dry transfer of proteins, a sandwich of Whatman paper / gel / membrane / Whatman paper (wetted with the transfer buffer) is placed directly between cathode and anode in a blotting chamber (Peqlab, Biotechnologie GmbH, Erlangen, Germany cat. no. 52-2020). The blot was performed at 450 mA for 1 h. To prevent unspecific binding of antibodies, the membrane was incubated in block ing solution (2 % milk in PBST) at room temperature for 1 h at shaking conditions. Afterwards, the membrane was incubated in PBST 1 % milk supplemented with the primary antibody at room temperature for 1 h. Followed by washing the membrane 3 x with PBST for 15 min, the membrane was incubated with PBST 1 % milk containing a horse radish peroxidase (HRP) conjugated secondary antibody at room temperature for 1 h. After 3 x washing with PBST for 10 - 15 min, the blot was incubated with 1 ml Western Blotting Detection Solution and detection of the chemiluminescence, emanating from the HRP-mediated conversion of the substrate was performed in an image reader .

June

5. June

Seeding cells for PEI transfection

The seeding of the cell was done as mentioned above

6. June

PEI transfection and coating of ELISA plate

The seeding of the cell was done as mentioned above

7. June

Testing the best oligonucleotide length for Cas9/crRNA binding

The three different biotinylated and annealed oligonucleotides oIG9000 / oIG9001, oIG9002 / oIG9003 and oIG9004 / oIG9005 (10 pmol / well) were coated by incubation at RT for 1 h. The transfected HEK-293T cells (250.000 cells / ml) were harvested by sonification for 10 minutes in the dilution buffer (10 mM Tris, 1 % BSA, 125 mM NaCl, 10 mM MgCl2) and (10 mM Tris, 1 % BSA, 50 mM MgCl2, 10 mM NaCl) containing one tablet of the EDTA free Protease inhibitor cocktail per 50 ml buffer. The Buffer dilution comprising the Cas9 were performed in round 96-well plates by a 1:1.

NaCl [mM]	MgCl2 [mM]
125	50
125	25
125	12.5
125	6.25
125	10
10	50

The results show that a total length of 40 nucleotides is not enough for the Cas9 / RNA to bind. 50 nucleotides are sufficient for binding however 60 nucleotides show the most robust binding behaviour

12. June

Seeding cells for PEI transfection

The seeding of the cell was done as mentioned above

13. June

PEI transfection and coating of ELISA plate

The PEI transfection and coating of the ELISA plate was done as mentioned above

14. June

Optimizing the buffer condition

different concentrations of MgCl2 – 30 mM, 20 mM, 10 mM and 5 mM – and of NaCl – 150 mM, 120 mM, 90 mM and 60 mM, were varied against each other.

NaCl [mM]	MgCl2 [mM]
150	30
150	20
150	10
150	5

NaCl [mM]	MgCl2 [mM]
120	30
120	20
120	10
120	5

NaCl [mM]	MgCl2 [mM]
90	30
90	20
90	10
90	5

NaCl [mM]	MgCl2 [mM]
60	30
60	20
60	10
60	5

The ELISA was performed as explained above. Dilution buffers containing (10 mM Tris, 1 % BSA, NaCl – 150 mM, 120 mM, 90 mM and 60 mM, MgCl2 – 30 mM, 20 mM, 10 mM and 5 mM respectivly) containing one tablet EDTA free Protease inhibitor cocktail per 50 ml were used to uptake the transfected HEK – 293T cells and subsequently perform cell lysis.

The figure shows that the binding behaviour of the Cas9 / crRNA towards EMX1 display similar behaviour for descending MgCl2. This is true for all four NaCl concentrations however 150 mM NaCl has the smallest yield.

Labbook Cas9-Truncation

July

23. July

PCR Cas9

µl	type
10	Q5-HF Reaction Buffer
1	Template: pIG2004
1	oIG9100
1	oIG9101
4	dNTPs
1	DMSO
0,5	Q5-HF Polymerase
Add to 50	H₂O

Annealing: 60°C
Extension: 2min20sec
18 cycles

Roth 1kb ladder - Cas9-PCR a - Cas9-PCR b - 2log ladder NEB
There is a second (unspecific?) band in b.

Gel extraction of PCR Cas9

Bands a and b were cut out and extracted separately with Roche high pure PCR product purification kit. Eluation in 20 µl H₂O, before centrifugation: incubated 10min at room temp. Eluted liquid put on column again, incubated for 10 min and centrifuged. concentrations: less than 5ng, no digest performed. -> repeat PCR tomorrow.

24. July

PCR Cas9

PCR was repeated with same program but 25 cycles instead of 18. This time no unspecific bands were visible (picture could not be saved). Both bands were cut out and extracted over same column with Roche Kit.
Concentration: 65 ng/µl

Digest with SacII and KpnI-HF

µl	type
10	pIG6000 (170ng/µl=1.7µg)
2µl	NEB-Buffer Cut-Smart
1µl	SacII
1µl	KpnI-HF
Add to 20 µl	H₂O

µl	type
25	Cas9-PCR (65 ng/µl=1,625µg)
5µl	NEB-Buffer Cut-Smart
1µl	SacII
1µl	KpnI-HF
Add to 50 µl	H₂O

Temp.: 37°C
Incubation time: 1.5h

0.7% agarose gel, upper bands cut out, digest worked.

Roth ladder - Cas9-KRAB (4826bp) - pIG6000 (upper band 2721bp, insert 1064bp)

Gel extraction of digested fragments

Cas9: 16.2ng/µl
dIG6000: 17.5ng/µl
frozen at -20°C

25. July

Ligation of digested Cas9-PCR product and dIG6000

Calculation:

Cas9-PCR = backbone (4826bp)
dIG6000 = insert (2721bp)

(30ng backbone/bp backbone) x bp insert x 3 = ng insert
30ng Cas9-PCR/4826 x 2721 x 3 = 50.7ng dIG6000

amount	substance
30ng = 1.85µl	Cas9-PCR (16.2ng/µl)
50.7ng = 2.89µl	dIG6000 (17.5ng/µl)
2µl	T4 ligase buffer (Fermentas)
1µl	T4 Ligase (Fermentas)
Add to 10 µl	H₂O

30min at 22°C

Transformation

2.5µl of ligation used. >1h on 37°C shaker.
plate 50µl, centrifuge & discard supernatant, plate rest.
2 a.m.: colonies picked for mini prepping.

26. July

Miniprep of colonies 1-4

with Roche kit.

Mini	1	2	3	4
concentration in ng/µl	77.7	13.9	65	45

Testdigest of Minis with SacII and KpnI

µl	type
about 200 ng	DNA
1	NEB-Buffer Cut Smart
0.25	SacII (undiluted)
0.5	KpnI-HF
Add to 10 µl	H₂O

Temp.: 37°C
Incubation time: 1-2h
expected fragments:

4826bp for Cas9-KRAB-bgh
2722bp for pSB1C3-CMV (dIG6000)

Roth marker, mini1, 2, 3, 4, pIG6000
Cas9-KRAB (4826bp) pIG6000 (upper band 2721bp, insert 1064bp)
in mini2 slight Cas9 band is visible (marked by star).

Concentration too low to send for sequencing, try PCR with truncation primers instead. If Cas9 is there, PCR should work.

27. July

Test-PCR on Mini2 for Truncation 2

fw: oIG9032 rev: oIG9033
fragment size: 7250bp

Annealing: 63°C
Extension: 4min
25 cycles

Mini preps of ligation colonies 5-7 and 2&4 again to yield higher concentrations

Mini	2	4	5	6	7
concentration in ng/µl	193.1	181.9	230.8	171.8	215.8

Test digest of Minis with SacII and KpnI

For protocol see 26.7.13. 1µl of each Mini prep was used.

0.7% Agarose gel of PCR and Test Digest

up: Roth 1kb ladder - Test PCR1 - Test PCR2
down: Roth 1kb ladder - Mini2 - 4 - 5 - 6 - 7
4, 5 and 7 seem to be positive!

Roth 1kb ladder - Mini2 - 4 - 5 - 6 - 7
Mini 5 is not completely digested, probably because of high concentration (230ng/µl!)

Minis 5 and 7 contain Cas9-KRAB and can be used as truncation PCR templates.

29. July

Truncation PCRs

annealing: 64°C
elongation: Truncation 1&2 4min, Truncation 3,4 & 5 3:30min

Roth 1kb ladder - Truncation1 - 2 - 3 - 4 - 5

Gibson of truncated Cas9 plasmids

Trafo of Gibson

30. July

Truncation 3 and 4 yielded 10-15 colonies each. No colonies on other plates. 8 colonies from each plate were picked and streaked out for mini preps.

Truncation PCR 1, 2 and 5 repeated as on 29.7.

Roth 1kb ladder - Truncation PCR 1 - 1 - 2 - 2

Roth 1kb ladder - Truncation PCR 5 - 5 - BbsI digested pIG3010

BbsI digest of RNA plasmid (from Hormon group)

Roth 1kb ladder - Truncation PCR 5 - 5 - BbsI digested pIG3010

31. July

Mini preps and test digest of truncation 3 and 4

Minis: >200ng/µl, 1µl was digested with EcoRI and SpeI (both HF) for 2h at 37°C.

Roth 1kb ladder - MiniT3 1-2-3-4-5-6-7-8- -pIG9100
Roth 1kb ladder - MiniT4 1-2-3-4-5-6-7-8- - gelex PCR1-2-5- Roth ladder

Gel Ex of truncation PCR 1, 2 and 5

9-15ng/µl, test size on gel.

gelex PCR1-2-5- Roth ladder

Gibson of truncation 1, 2 and 5

5µl of gel extracted PCR product were added to aliquoted Gibson master mix, 1h 50°C. 300µl were plated on chloramphenicol plates.

Testdigest

as the sequenzing of T3 showed unclear results we did minipreps of 4 more colonies from the gibson plate and digested them with EcoRI and SpeI to yield another positive testdigest that could be sequenzed.

RNA-Plasmid

Gel ex of BbsI digested plasmid

Oligo annealing of EGFP oligos

Ligation with EmxI and EGFP crRNA target oligos

August

6.8.

Seeding cells for transfection

120000cells/ml were seeded in six well plates (3ml per well)

Sequencing of RNA Plasmids

Both EGFP and EMX1 were inserted correctly (enter clone number)

7.8.

Inoculate Midis for T1, 2, 5, pIG9100(Cas9), pIG3010(EMX1, EGFP)

100ml LB + 100µl Chloramphenicol. For T2 four additional minis were inoculated for midi and sent for overnight sequencing.

Truncation 4

Due to 200 missing base pairs more colonies could be screen from T4 plate: 9 clones streaked out for mini prepping (edit 8.8 plates put in 4°C bacterial fridge, prep when suitable).

8.8.

Midipreps of plasmids

All plasmids were prepped because sequencing was delayed. Concentrations: ?

Transfection

Medium change was performed 7 hours before transfection.

3µg DNA were transfected per well (six well plate). Of truncation 2 all midis were transfected because sequencing results were still missing (pIG9102-3, -1, -5, -7 and -8 transfected). Transfection scheme?

ELISA

96 well plate (flat, high-binding) was coated with streptavidin (100µl per well, 4µg/ml working solution), sealed and incubated over night at 4°C.

10xTBS was prepared (50mM Tris, 125mM NaCl, pH 7.5)

9.8.

Cell lysis and ELISA (Fenja and Philipp)

did not work; repeat everything.

12.8.

Cell Seeding

x cells/ml seeded in 6 well plate

Sequencing Results for T2

T2.3 is positive

13.8.

Transfection

Natalie, scheme

ELISA II

Coating plate

14.8.

Cell lysis

with dilution buffer (500µl per 6-well) and sonification (10min)

ELISA II

loading scheme?
ELISA did not work. Secondary antibody broken? Try again..

15.8.

Cell Seeding

16.8.

Transfection

PIF-GFP as transfection control

17.8.

ELISA III

no Cas9 in pSB1C3 detectable. Test if Cas is expressed: Western Blot with remaining cell lysat against HA-tag.

18.8.

Cell lysis and Blot

Done at AG Warscheid. anti-HA antibody overnight.

19.8.

Detection of blot

anti-mouse antibody (xh), washing steps, detection with ECL. illumination for 1000sec, only unspecific bands. Repeat western Blot: transfection, lysis, SDS PAGE, blot.

Cell seeding and transfection

HEK293-H from Adrian were seeded and transfected 5?h afterwards.

20.8.

Transfection did not work. Seed new cells.

21.8.

Transfection

let cells grow for 48h! Medium change after 5h.

22.8.

SDS gels

23.8.

Cell Lysis

with modified RIPA buffer from Adrian (200µl per 6-well), incubated on ice 10min, scraped off with pipet tip, in eppi, on ice for 5-10min, centrifuge 5min, 10000xg.
boil 150µl lysate with 50µl 4x sample buffer (5min, 95°C)

SDS PAGE

Western Blot

anti-HA mouse 1:2000 (AG Weber) in TBS with BSA.

24.8.

detection

secondary antibody (1.5µl anti-mouse in 25ml TBS), wash for 2h..

still no cas9 in pSB1C3!

25.08-28.08.13

Natalie: Cas9-mCherry?

29.08.13

Truncation PCRs with standardized Cas9 (CMV-HA-NLS-Cas9-bGH)

all PCRs were done as on 29.07.13 with 4min elongation time and 64 °C annealing temperature. All PCRs worked.

Truncation PCRs T1, T2, T3, T4, T5

Gel extraction of PCR products

with Roche kit, eluted in 25?µl H₂O. Concentrations between 10-40ng/µl.

Gibson and transformation of standardized truncations

5 µl of PCR product (gelex) were mixed with Gibson mastermix, 1h 50°C, 3min RT, 3min on ice, trafo with 5 µl. Everything plated.

30.08.13

Picking colonies and minipreps

colonies for all truncations were picked and inoculated for mini-prepping in liquid cultures. Mini preps were not successful because cultures were too thin. more colonies were streaked out on agar plates to grow over night.

31.08.13

Mini Preps and test digest of standardized truncations

four colonies of each T1, T2, T3, T4, T5 were preped with Roche kit and eluted in 50 µl H₂O.

Mini preps were test digested with XbaI and PstI-HF in Cut-Smart buffer (10 µl volume, 2h, 37 °C) and separated on a 1% agarose gel.

31.08.13 test digest. Log2 marker, T1-1,-2,-3,-4, T2-1, etc.

Seeding Cells

2/3 of one 10cm plate were seeded into two 6well plates.

Midi preps of standarized truncations

Midi preps of pIG9200-9205 (chloramphenicol) + pIGCas-mCherry (ampicillin) were inoculated in 100 ml LB+100 µl antibiotic (Mini T1-1, T2-1, T3-1, T4-3, T5-1).

September

01.09.13

Midi preps of standardized truncations

Plasmids were midi prepped with Promega vacuum "Sau", 6ml buffer amounts used, columns were not dried from ethanol before eluting. Some columns were clogged and had to be scraped free with steril pipet tips. Plasmids were eluted in 300 µl nuclease free H₂O. Where possible Midis were diluted to 400ng/µl.

Plasmid	pIG9200	pIG9201	pIG9202	pIG9203	pIG9204	pIG9205	pIGCas9-mCherry
concentration in ng/µl	400	400	400	400	400	240	400

Transfection of standardized truncations

All midi-prepped plasmids were transfected (6well protocol, 3µg total DNA) with pIG9000, PIF-GFP and pIG2004 as controls. This transfection will be used for western blotting to see if the standardized Cas9 is expressed in pSB1C3.

02.09.13

Sequencing

Midi preps of standarized truncations 1-5 were sent for sequencing with CMV forward primer @ GATC or oIG6018.

Cells seeded for ELISA

65000 cells/well were seeded in 6-well plates as transfection will only be on wednesday (04.09.).

03.09.13

Sequencing results and cell harvesting

All sequencing results were useless (sequence length 0nt). Therefore no western blot was done today but cell pellets were collected and frozen. wells transfected with fluorescent proteins were photographed (link pics..). For harvesting cells were washed with cold PBS and then rinsed from well bottom with 500 µl cold PBS and collected in 1.5ml Eppis. Samples were centrifuged 5min at full speed (21000xg), supernatant was decanted and cell pellets were frozen at -20°C.

pIG9200 and mini T1-1 (pIG9201) were sent for sequencing again with CMV-F and BGH-R (both @ GATC).

04.09.13

Western blot

still no sequencing results. pellets blotted anyway. sample buffer, sonifyer, SDS PAGE, semidry blot (not enough transfer buffer?? gels dried out), block in TBST+milk 1h30, anti-HA-mouse 1:2500 in TBST+milk over night.

GATC is repeating seuquencing of pIG9200 and pIG9201 with oIG6017.

retrafo and new minis (in 1.5ml eppis) of T1-5? Natalie.

05-07.09-13

Sequencing results

sequencing did not work because no CMV promoter in construct! finally sequenced with oIG6017: result: SV40(!)-NLS-Cas-BGH, no CMV promoter and no HA-tag! Therefore all our truncation constructs are useless. We have to wait for the standardized CMV-HA-NLS-Cas9-BGH construct.

09.09.13

Cloning of CMV-HA-NLS-Cas9-BGH

We received HA-NLS-Cas9-BGH from standardization group and have to ligate it with the CMV promoter.

Mini prepping and test digest

some colonies were prepped and test-digested with NgoMIV or NotI.(Natalie)
Mini1 (407ng/µl) was used for digest.

Digestion with EcoRI/XbaI and EcoRI/SpeI

HA-NLS-Cas9-BGH is cut open with EcoRI-HF and XbaI, the CMV promoter (pIG0019, 5ng/µl) is cut out with EcoRI-HF and SpeI-HF to then be ligated into the opened Cas9-backbone.

µl	type
1-2µg (4µl Cas9, 20µl CMV)	DNA
5	Cut-Smart NEB buffer
1	EcoRI-HF
1	XbaI / SpeI-HF
Add to 50µl	H₂O

Temp.: 37°C
Incubation time: 2h

Roth 1kb marker - HA-NLS-Cas9-BGH - CMV
Cas9 backbone has correct size (6466bp), CMV insert (603bp) maybe lies in bromphenol blue band? Cut out and gelex anyway.

Gelex

both bands were gelexed with Roche Kit and eluted in 20µl H₂O.

HA-NLS-Cas9-BGH = 38,9ng/µl
CMV = 5ng/µl

Ligation

50ng of Cas9 backbone were ligated with 15ng of CMV insert. Two ligations were set up, with our CMV-digest (N+L) and one of the standardization group (St.)

µl	type
1.5	Cas9 backbone
3	CMV insert (N+L or St.)
2	T4 buffer
1	T4 Ligase (Fermentas?)
12.5	H₂O

Room temperature
Incubation time: 30min

Trafo

4µl were used for trafo of each ligation

10.09.13

Minis

four colonies of each ligation were picked and inoculated in 1.5ml Eppis with 1ml LB+ 1µl Chloramphenicol at 37°C, 700rpm. Eppis were opened frequently to ensure O₂ supply.

11/12.09.13

Sequencing results

no CMV promoter...

12-18.09.13

New cloning strategy with new CMV promoter

Ligations done again to produce SV40-HA-NLS-dCas9-NLS-BGH (pIG0063) and CMV-HA-NLS-dCas9-NLS-BGH (pIG0062). Both used as templates for truncation PCRs. Only CMV constructs shall be tested at first, as we expect higher expression (and thus more easily detectable binding levels in uniBAss) here.

19-20.09.13

Sequencing of truncation minis

Minis for each truncation with CMV promoter were sent for sequencing, had to be repeated for T1,2,4 and 5 due to frameshifts/extra primer insertions etc.. Wait for results on Monday, but inoculate midis anyway on Sunday.

22.09.13

Seeding cells

4 6well plates were seeded with 130000cells/ml=260000cells/well.

Midis inoculated

Midis of T1-T5 were inoculated (except for T3, already done by Michi and Nadine). Gabi :)

23.09.13

Midis and transfection of CMV-T1-5

Finally sequencing results were correct (T4 was repeated:check tomorrow), inoculated midis were prepped with Promega kit (double buffer amounts) and eluted in 600µl nuclease free H₂O.

Transfection was performed as on plan in 6well plates. Additionally all standardized constructs (CMV and SV40) and their non-standardized pX334a-versions were transfected to be compared on uniBAss. The Assay will be performed 48h after transfection. change of media required? check in the morning!

24.09.13

No medium change of the cells transfected the day before was performed. SDS-Gel for the westernblot which will be performed tomorrow poured. the ELISA plate for uniBAss is coated with strepdadivin.

25.09.13

uniBAss Day

The HEK cells were lysed about 42h after transfection. the uniBAss ELISA was performed according to the protocol. in parallel a westernblot was performed with 2/5 of the lysate of each well to ensure Cas9 expression.

25.09.13

westernblot of uniBAss probes

the westernblot of yesterday was analyzed. Due to problems with resolving the protein pellet we had suboptimal results in the blot. therefore we want to repeat the hole experiment. as two days before we transfected HEK cells in 6-wells. this time we made biological duplicates for the standardised constructs. (together six 6-wells)

26.09.13

Medium of the six 6 wells was exchanged 12h after transfection.

27.09.13

westernblot of 2.uniBAss probes

As an expression control we wanted to perform the westernbot of the transfected cells this time before we to the uniBAss ELISA. So we collected the cells in 500µl diluent buffer and took 170µl for western blotting. the 170µl cell suspension was centrifuged for 5min at 10000g, the supernateand decanted and the pellet resuspended in 100µl SDS-probe buffer. then the suspension was sonified for a total of 35min. the probes were cooked for 15min and SDS-page was performed, followed by a semidry westernblot. after blocking the 1.Ab (mouse-anti-HA) was applied over night.

28.09.13

development of the westernblot. after washing and applying the secondary antibody we developed the membranes. we could detect most of our constructs and had very nice bands. so we want to perform the uniBAss with all our probes tomorrow, therefore we coated an ELISA plate with streptavidin.

29.09.13

uniBAss Day...agian...

to perform the uniBAss we had to unfreeze our probes from the -80degree freezer. then we sonifyed them and proceeded with our usual uniBAss-protocol.

@@ Line 1,573: / Line 1,573: @@
    <h2> 25.09.13 </h2>
    <h3> uniBAss Day</h3>
-   <p> The HEK cells were lysed  about 42h after transfection. the uniBAss ELISA was performed as in the protocol. in parallel a westernblot was performed with 2/5 of the lysate of each well to ensure Cas9 expression.
+   <p> The HEK cells were lysed  about 42h after transfection. the uniBAss ELISA was performed according to the protocol. in parallel a westernblot was performed with 2/5 of the lysate of each well to ensure Cas9 expression.
    </p>
+	<h2> 25.09.13 </h2>
+		<h3> westernblot of uniBAss probes</h3>
+			<p> the westernblot of yesterday was analyzed. Due to problems with resolving the protein pellet we had suboptimal results in the blot. therefore we want to repeat the hole experiment. as two days before we transfected HEK cells in 6-wells. this time we made biological duplicates for the standardised constructs. (together six 6-wells)  </p>
+</div>
+<div id="tag">
+	<h2> 26.09.13 </h2>
+	<p>Medium of the six 6 wells was exchanged 12h after transfection. 	  </p>
 </div>
+<div id="tag">
+	<h2> 27.09.13 </h2>
+	<h3> westernblot of 2.uniBAss probes</h3>
+	<p> As an expression control we wanted to perform the westernbot of the transfected cells this time before we to the uniBAss ELISA. So we collected the cells in 500&micro;l diluent buffer and took 170&micro;l for western blotting. the 170&micro;l cell suspension was centrifuged for 5min at 10000g, the supernateand decanted and the pellet resuspended in 100&micro;l SDS-probe buffer. then the suspension was sonified for a total of 35min. the probes were cooked for 15min and SDS-page was performed, followed by a semidry westernblot. after blocking the 1.Ab (mouse-anti-HA) was applied over night. </p>
+</div>
+<div id="tag">
+	<h2> 28.09.13 </h2>
+	<p> development of the westernblot. after washing and applying the secondary antibody we developed the membranes. we could detect most of our constructs and had very nice bands. so we want to perform the uniBAss with all our probes tomorrow, therefore we coated an ELISA plate with streptavidin.</p>
+</div>
+<div id="tag">
+	<h2> 29.09.13 </h2>
+	<h3> uniBAss Day...agian...</h3>
+	<p> to perform the uniBAss we had to unfreeze our probes from the -80degree freezer. then we sonifyed them and proceeded with our usual uniBAss-protocol. </p>
+</div>
 </div>
 </body>
 </html>