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Team:Freiburg/Notebook/method
From 2013.igem.org
Labbook Cas9-Truncation
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:
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
| 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
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).
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. ?? 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!
