Team:Freiburg/Notebook/method

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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

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:
      1. 4826bp for Cas9-KRAB-bgh
      2. 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 as in the protocol. in parallel a westernblot was performed with 2/5 of the lysate of each well to ensure Cas9 expression.

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