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Team:Freiburg/Notebook/lab epigenetics
From 2013.igem.org
05.06.13
G9a mus musculus DNA arrived from Stuttgart
Lyophilized DNA on Whatman paper was incubated with 50µl sterile H2O (10min at RT) and eluted from the paper by centrifugation: 51.7ng/µl (NanoDrop).
20µl were sent for sequencing.
Transformation of G9a
4µl of G9a-DNA were transformed into 25µl Top10 competent E. coli. 250µl LB medium was added and cells were grown on 33°C for 1h30min before plating on an ampicillin agar plate.
06.-07.06.13
Clone selection
Because no single clones could be picked, a dilution plating of the transformation was performed and two single clones were selected the next day and streaked out for mini preps.
08.06.13
Mini preps of G9a clones
Clones were prepped with the Roche High Pure Plasmid Isolation Kit. Both clones were sent for sequencing with oIG8007 and oIG8008.
| Clone | 1 | 2 |
|---|---|---|
| concentration in ng/µl | 61 | 76 |
09.06.13
Sequencing results
Both clones contain G9a from mus musculus.
New primers to amplify G9a(mm) were designed and ordered.
insert primer list here?
14.06.13
New primers arrived (oIG8009-oIG8012)
PCR to amplify G9a-SET domaine
aIG8000 (linker-G9a-NLS): oIG8009 (fw) and oIG8010 (rev) with G9a-Mini prep 1 (8.6.) as template.
aIG8003 (NLS-G9a-linker): oIG8011 (fw) and oIG8012 (rev).
| µl | type |
|---|---|
| 10 | Q5-HF Reaction Buffer |
| 1 | Template |
| 1 | Primer1 |
| 1 | Primer2 |
| 4 | dNTPs |
| 1 | DMSO |
| 0.5 | Q5-HF Polymerase |
| Add to 50 | H2O |
- Annealing: 60°C
- Elongation: 30 sec
- 24 cycles
| |
| PCR aIG8000 and aIG8003 |
Bands run too low, can be due to gelred. Check size after gelextraction by loading less DNA.
Gel extraction of PCR products aIG8000 and aIG8003d
| name | ng/µl |
|---|---|
| aIG8000 | 115.6 |
| aIG8003 | 105.4 |
PCR to amplify Cas9 (on pX334a)
We need the mutated non-cleaving Cas9. Therefore we use pIG2004 from Max as template to produce aIG8001 (oIG2000/oIG8002) and aIG8002 (oIG2006/oIG2009).
5µl of Q5 buffer were used instead of 10µl. Elongation: 4min20sec.
| |
| PCR aIG8001, Gelex aIG8003(size correct), marker, aIG8002 |
Gel extraction of PCR products aIG8001 and aIG8002
| name | ng/µl |
|---|---|
| aIG8001 | 19.2 |
| aIG8002 | 11.5 |
15.06.-05.08.13
waiting to be digitalized...
6.8.
SDS
Semidry Blot
Western Blot
7.8.
Western Blot of G9a continued
First antibody was decanted (and stored for further use at -20°C) and membrane was washed 3x 15 min. Secondary antibody (anti-mouse with HRP) was diluted 1:5000 in 2% milk powder in PBS and incubated in foil sealed for 2h. Antibody was decanted and membranes were washed with PBST (?%Tween in 1x PBS) in boxes (without foil) 3x for 5min.
Chemiluminescent detection of HRP
With image Quant: Membrane was placed on foil (in middle), focus adjusted. ECL I & ECL II were mixed (600 µl each) and spread over membrane (no incubation time). Imaging was started right away. Settings: Auto (overexposed). Measurement of 1a-HA was repeated with 1min exposure time.
16.08.13
Red light inducible PhyB-G9A and Cas9-PIF system
PCR amplification of the backbone containing PhyB for Gibson
| µl | type |
|---|---|
| 10 | Q5-HF Reaction Buffer |
| 1 | pKM018 (30ng) |
| 1 | oIG8021 |
| 1 | oIG8022 |
| 2.5 | dNTPs |
| 1.5 | DMSO |
| 0.5 | Q5-HF Polymerase |
| Add to 50 | H2O |
- Annealing: 60°C
- Extension: 2min 30sec
PCR amplification of G9A with specific overhangs to the PhyB-bb
| µl | type |
|---|---|
| 10 | Q5-HF Reaction Buffer |
| 1 | pIG8002 |
| 1 | oIG8023 |
| 1 | oIG8024 |
| 2.5 | dNTPs |
| 1.5 | DMSO |
| 0.5 | Q5-HF Polymerase |
| Add to 50 | H2O |
- Annealing: 60°C
- Extension: 40sec
Results
 |
| Left side: 2log laddder; On the right side: G9A; everything else are different probes |
 |
| Left: 2log ladder, right: PhyB bb |
All bands had the expected size (gelred standard) and were band isolated and purificated
19.08.13
Red light inducible PhyB-G9A and Cas9-PIF system
A Gibson was performed using the PCR products of PhyB-bb and G9A in a ratio of 1/8 (58.39 ng PhyB; 0.9 43.25 ng G9A). The control plate showed nearly as many colonies and a test digest using Hind3 and EcoR1 only revealed one possitive colony. The sequencing showed a frameshift therefore a Gibson has to be repeated by using another ratio (maybe 1/4).
22.08.13
Red light inducible PhyB-G9A and Cas9-PIF system
Another gibson approach of G9A PhyB-bb using a 1/4 ratio
Gibson approach:
Test digest of some Gibson colonies using Sal1 and EcoR1
 |
| Left: 2 log ladder; then tested Gibson colonies from 1-10. Colony 5-10 seem to be defenitly possitive. |
Colony 5 was send for sequencing
Ligation of two VEGF crRNAs into pIG3010
For light experiments a plasmid containing the tracrRNA and crRNA is needed.
Ligation
| µl: | Substance: |
|---|---|
| 1 | pIG3010 |
| 5 | crRNA2/3 (2/3 refere to the different VEGF target sides) |
| 2 | 10X T4-Ligase Buffer |
| 1 | T4-Ligase |
| 11 | H2O |
| 20 | Total |
- 0.5h at RT
27.08.13
Red light inducible PhyB-G9A and Cas9-PIF system
Sequencing results of pIG8007 (PhyB-G9A) - the sixed colony from test digest was send for sequencing
A frameshift is inside the sequencing. Colonies 9 and 10 were send for sequencing and prepared for midi prep
crRNA-tracr Plasmid with two VEGF target sides
Colonies of crRNA ligation were minipreped and 2 colonies of each locus (2.1 2.2 3.1 3.2) were send for sequencing and emediatly prepared for minipreps
First cellculture experiment
Two 24 well plates were prepared with HEK293T cells (65,000 cells/well).
