Team:Freiburg/Notebook/lab epigenetics

From 2013.igem.org

(Difference between revisions)

Revision as of 22:13, 14 September 2013

Effector - Epigenetics

June

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

August

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).

@@ Line 46: / Line 46: @@
 <p class="second_order"> <a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_repression">Repression </a> </p>
 <p class="second_order"> <a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_epigenetics">Epigenetics </a> </p>
+<p class="third_order"> <a href="#june"> June </a> </p>
+<p class="third_order"> <a href="#august"> August </a> </p>
 <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/induction"> Induction </a> </p>
@@ Line 66: / Line 69: @@
 <div id="h1">
 Effector - Epigenetics
+</div>
+<div id="june">
+<p id="h2">
+June
+</p>
 </div>
@@ Line 105: / Line 114: @@
 	</div>
 	<div id="tag">
@@ Line 236: / Line 246: @@
 	</div>
+<div id="august">
+<p id="h2">
+August
+</p>
+</div>
 	<div id="tag">