Team:Freiburg/Notebook/method

• Annealing: 60°C
• Extension: 2min20sec
• 18 cycles

• Temp.: 37°C
• Incubation time: 1.5h

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

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

Testdigest of Minis with SacII and KpnI

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

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

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.

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.

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.)

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. ?? Gabi

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!