Team:Freiburg/Notebook/lab repression
From 2013.igem.org
(14 intermediate revisions not shown) | |||
Line 40: | Line 40: | ||
</p> | </p> | ||
+ | <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/crrna"> Targeting </a></p> | ||
<p class="first_order"><a class="active" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_effector"> Effector </a></p> | <p class="first_order"><a class="active" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_effector"> Effector </a></p> | ||
- | <p class="second_order_note"> <a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_activation">Activation </a> </p> | + | <p class="second_order_note"> <a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_activation"> Activation </a> </p> |
- | <p class="second_order_note"> <a class="active" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_repression">Repression </a> </p> | + | <p class="second_order_note"> <a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_epigenetics"> Epigenetics </a> </p> |
+ | <p class="second_order_note"> <a class="active" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_repression"> Repression </a> </p> | ||
<p class="third_order"> <a href="#april"> April </a> </p> | <p class="third_order"> <a href="#april"> April </a> </p> | ||
Line 51: | Line 53: | ||
<p class="third_order"> <a href="#september"> September </a> </p> | <p class="third_order"> <a href="#september"> September </a> </p> | ||
- | + | ||
- | <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/induction"> | + | <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/induction"> Effector Control </a> </p> |
- | + | ||
- | + | ||
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/modeling"> Modeling </a></p> | <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/modeling"> Modeling </a></p> | ||
- | <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/standardisation"> | + | <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/method"> uniBAss </a></p> |
- | <p class="first_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/protocols"> | + | <p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/standardisation"> Standardization </a></p> |
+ | <p class="first_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/protocols"> Material and Methods </a> </p> | ||
Line 70: | Line 73: | ||
<div id="h1"> | <div id="h1"> | ||
- | Effector - Repression | + | Effector - Repression - KRAB |
</div> | </div> | ||
Line 2,000: | Line 2,003: | ||
<table class="gelpic"> | <table class="gelpic"> | ||
<tr> | <tr> | ||
- | <td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/ | + | <td> <img class="gelpic"; src="https://static.igem.org/mediawiki/2013/6/62/Freiburg_GFP_Reprimierung_3_09-1.jpg"> </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> Results of GFP repression. Experiment | + | <td> Results of GFP repression. Fluorescence images were taken with a light exposure time of 250 ms (80% saturation). Experiment shall be repeated with off-target controls and parallel Flow Cytometry analysis. </td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 2,273: | Line 2,276: | ||
</ul> | </ul> | ||
+ | <div> | ||
+ | <table class="imgtxt" width="600px"> | ||
+ | <tr> | ||
+ | <td> <img class="imgtxt" width="600px" src="https://static.igem.org/mediawiki/2013/8/8a/Western_Blot_II_Freiburg_2013.PNG"> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> <b>Western blot of the SEAP assay from 23.09. </b><br> | ||
+ | CAG:dCas was strongly expressed, whereas CMV:dCas and dCas-KRAB were not visible on the western blot. Nevertheless a effect has been detected with the SEAP assay, so there should be an expression, but maybe too low for detection. | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | |||
+ | <h2>27.09.13</h2> | ||
+ | <h3>Medium removal</h3> | ||
+ | <ul> | ||
+ | <li>Supernatant was collected for SEAP measurement 48 h after transfection.</li> | ||
+ | <li>Cells were frozen at - 80 °C for later on Western blotting.</li> | ||
+ | </ul> | ||
+ | |||
+ | <h2>28.09.13</h2> | ||
+ | <h3>SEAP measurement</h3> | ||
+ | <ul> | ||
+ | <li>Collected supernatant and fresh cell culture medium was heated at 65 °C for 30 min, then centrifuged at 1250 g for 1 min.</li> | ||
+ | <li>Supernatant of wells containing CMV:SEAP was diluted in cell culture medium 1:15 (6.7 µl supernatant + 93.3 µl medium).</li> | ||
+ | <li>80 µl of (diluted) supernatant was given to 100 µl of SEAP buffer.</li> | ||
+ | <li>After addition of 20 µl pNPP (substrate of SEAP) the absorbance of the mix was measured every minute for 2 h at 405 nm. | ||
+ | </ul> | ||
+ | |||
+ | <h3>Cell lysis for lyciferase measurement and western blot</h3> | ||
+ | <ul> | ||
+ | <li>250 µl of lysis buffer (containing Tris-HCl, EGTA, MgSO<sub>4</sub>, DTT and protease inhibitor) were applied to the thawn cells of each well.</li> | ||
+ | <li>Incubation on ice for at least 10 min.</li> | ||
+ | <li>Centrifugation at 2,200 g for 30 min.</li> | ||
+ | </ul> | ||
+ | |||
+ | <h3>Luciferase luminescence measurement</h3> | ||
+ | <ul> | ||
+ | <li>80 µl of supernatant were pipetted in a white 96 well plate.</li> | ||
+ | <li>Measurement of luminescence (every 2 min for 15 min) immediately after addition of 20 µl Renilla substrate in PBS.</li> | ||
+ | </ul> | ||
+ | |||
+ | <div> | ||
+ | <table class="imgtxt" width="700px"> | ||
+ | <tr> | ||
+ | <td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/parts/d/d0/Repression_SEAP_III_Freigem2013.png | ||
+ | "> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> <b>SEAP-Assay: Results </b><br> | ||
+ | All different tested loci showed no clear result. dCas9-KRAB under the control of the CMV promotor showed no significant repression in comparison to the off-target and CRISPRi control. dCas9-KRAB under the control of the SV40 promotor showed no repression at all. Maybe targeting a gene coded on a plasmid is a problem for dCas9-KRAB to repress. | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <h2>29.09.13</h2> | ||
+ | |||
+ | <h3>Protein precipitation</h3> | ||
+ | <p>As the cell lysis needed to be done with a high amount of lysis buffer, proteins are too much diluted for western blotting. Thus, they were precipitated:</p> | ||
+ | <ul> | ||
+ | <li>340 µl of acetone was added to 85 µl of the remaining lysates of one well per triplicate.</li> | ||
+ | <li>Incubation at - 20 °C for 1:15 h.</li> | ||
+ | <li>Centrifugation for 5 min at 10,000 g and 4 °C.</li> | ||
+ | <li>Removal of acetone and air drying for 5 min.</li> | ||
+ | <li>Addition of 25 µl 2x SDS-loading dye.</li> | ||
+ | <li>Heating at 95 °C for 5 min.</li> | ||
+ | </ul> | ||
+ | |||
+ | <h3>SDS-gel run</h3> | ||
+ | <p>SDS-gel was loaded with 20 µl of each sample. A voltage of 80 V was applied untill the loading dye bands reached the border between collection and separation gel, then the voltage was increased to 120 V.</p> | ||
+ | |||
+ | <h3>Western Blotting</h3> | ||
+ | <ul> | ||
+ | <li>Activation of PVDF-membrane for 10 min in methanol.</li> | ||
+ | <li>Short incubation of Whatman papers and the PVDF-membrane in transfer buffer.</li> | ||
+ | <li>Loading of western blot apperature: (-) - Whatman paper - SDS-gel - PVDF-membrane - Whatman paper - (+).</li> | ||
+ | <li>200 mA were applied for 1.5 h.</li> | ||
+ | </ul> | ||
+ | |||
+ | <h3>Antibody treatment I</h3> | ||
+ | <ul> | ||
+ | <li>Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 1 h.</li> | ||
+ | <li>Incubation with anti HA antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.</li> | ||
+ | <li>3 x washing with TBS-T for 5 min, each.</li> | ||
+ | <li>Incubation with anti mouse antibody for 1 h.</li> | ||
+ | <li>4 x washing with TBS-T for 10 min, each.</li> | ||
+ | <li>Measurement of luminescence after addition of ECL I + ECL II solution.</li> | ||
+ | <li>Air drying of the membrane for further antibody treatments.</li> | ||
+ | </ul> | ||
+ | |||
+ | <div> | ||
+ | <table class="imgtxt" width="400px"> | ||
+ | <tr> | ||
+ | <td> <img class="imgtxt" width="300px" src=" https://static.igem.org/mediawiki/parts/4/4d/Repression_SEAP_III_Western_Freigem_2013.png"> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> <b>Western blot of the SEAP assay from 28.09. </b><br> | ||
+ | Western blotting did not work well. Maybe lysis due to relative long incubation time on Renilla lysis buffer destroyed cells to much beforehand. So maybe only degradation products are visible. | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
</div> | </div> | ||
+ | </body> | ||
</html> | </html> |
Latest revision as of 05:15, 28 October 2013
April
21. April
Minipreps of pKM006, pKM102 & pSAM200
name | ng/µl |
---|---|
pSAM200 | 320.1 |
pKM006 | 113.3 |
pKM102 | 180.0 |
May
02. May
PCR of pX334 fragments
PCR 1
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2000
1
oIG2029
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 2
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2028
1
oIG2001
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 3
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2002
1
oIG2003
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 4
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2006
1
oIG2007
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 5
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2008
1
oIG2009
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | pX334 |
1 | oIG2000 |
1 | oIG2029 |
2.5 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | pX334 |
1 | oIG2028 |
1 | oIG2001 |
2.5 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 3
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2002
1
oIG2003
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 4
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2006
1
oIG2007
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 5
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2008
1
oIG2009
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | pX334 |
1 | oIG2002 |
1 | oIG2003 |
2.5 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | pX334 |
1 | oIG2006 |
1 | oIG2007 |
2.5 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 5
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2008
1
oIG2009
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | pX334 |
1 | oIG2008 |
1 | oIG2009 |
2.5 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
All samples were stored at -20°C
02. May
Agarose Gelelectrophoreses of PCR fragments No. 1-5
f.l.t.r.: marker, PCR 1 (1909 bp), PCR 2 (2610 bp), PCR 3 (1645 bp), PCR 4 (2272 bp), PCR 5 (1878 bp) |
Gel Extraction of PCRs 1-6
name | ng/µl |
---|---|
Fragment No. 1 | 40.7 |
Fragment No. 2 | 53.6 |
Fragment No. 3 | 52.6 |
Fragment No. 4 | 41.6 |
Fragment No. 5 | 40.4 |
03. May
Fusion PCR of PCR fragments 2-3 and 5-1
Assembly fragment 2-3 (PCR 1)
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | Fragment No. 2 |
1 | Fragment No. 3 |
4 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 71°C
- Extension: 105 sec.
- Cycles: 5
Assembly fragment 2-3 (PCR 2)
- Addition of 1 µl of oIG2028 and oIG2003 to PCR 1
- Annealing: 60 °C
- Extension: 2 min 40 sec
- Cycles: 16
Assembly fragment 5-1 (PCR 1)
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | Fragment No. 5 |
1 | Fragment No. 1 |
4 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 71°C
- Extension: 105 sec.
- Cycles: 5
Assembly fragment 5-1 (PCR 2)
- Addition of 1 µl of oIG2008 and oIG2029 to PCR 1
- Annealing: 60 °C
- Extension: 2 min 40 sec
- Cycles: 16
Gel Extraction of Fusion fragments 2-3 and 5-1
name | ng/µl |
---|---|
Fragment No. 2-3 (a) | 76.6 |
Fragment No. 2-3 (b) | 57.5 |
Fragment No. 2-3 (c) | 106.8 |
Fragment No. 5-1 | 23.4 |
17. May
PCR of the KRAB fragment with a N-terminal linker (GSAGSAG) overhang
PCR 1
µl
type
10
Q5-HF Reaction Buffer
1
pKM102
1
oIG2004
1
oIG2005
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 30 sec.
- Cycles: 18
- For higher Gel Extraction yields: 4x amount
Agarose Gelelectrophoreses of the KRAB fragment
f.l.t.r.: marker, KRAB at 490 bp (4x)
Gel Extraction of the KRAB fragment
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | pKM102 |
1 | oIG2004 |
1 | oIG2005 |
2.5 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 60°C
- Extension: 30 sec.
- Cycles: 18
- For higher Gel Extraction yields: 4x amount
f.l.t.r.: marker, KRAB at 490 bp (4x) |
name | ng/µl |
---|---|
KRAB (a) | 76.0 |
KRAB (b) | 64.7 |
Two bands were respectively pooled on one extraction column, in order to yield higher amount of DNA.
20. May
Gibson Assembly of pIG2004 (Cas9-KRAB)
- Preparation of 5µl DNA mix, containing all four fragments
- Melting a 15µl master-mix on ice, until it is ready to use
- Addition of 5µl of DNA mix to the master mix
- Immediately put the mix on 50°C and incubate for 1h
- 3 min on RT
- 3 min on ice
- Transformation of 4 µl to TOP10 chemically competent E-coli cells
Preparation scheme for Gibson Assembly fragment mix |
21. May
Evaluation of Gibson Assembly
- Colony growth
- Colony PCR will be performed with primers within KRAB and Cas9 fragment
Colony PCR Mastermix for Gibson Clones
µl
type
34.8
H2O
13.0
Taq Buffer
13.0
dNTPs
1.3
oIG2002
1.3
oIG2005
1.6
Taq Polymerase
5.0
H2O for each reaction, containing picked colony
- Annealing: 60°C
- Extension: 68°C
- Extension: 2 min 10 sec.
- Cycles: 30
Results of Colony PCR: Clones 5, 6 and 7 were striked out.
22. May
Miniprep of suspected pIG2004 Clones 5, 6 and 7
name
ng/µl
pIG2004, No. 5
207.9
pIG2004, No. 6
180.0
pIG2004, No. 7
198.1
Clones were sent to sequencing at GATC
23. May
Evaluation of Gibson Cloning Sequencing Results
oIG0007 sequencing reveals that the KRAB fragment was succesfully fused to Cas9 in each case. Further sequecing will be done to test for correct N-terminal assembly and H840A mutation.
PCR of pX334 fragment 6
PCR 1
µl
type
10
Q5-HF Reaction Buffer
1
pX334
1
oIG2002
1
oIG2007
2.5
dNTPs
1.5
DMSO
0.5
Q5-HF Polymerase
Add to 50
H2O
- Annealing: 60°C
- Extension: 2 min 40 sec.
- Cycles: 18
Agarose Gelelectrophoreses of PCR fragments No. 6
f.l.t.r.: marker, PCR 6 (3908 bp), empty, PCR 6 (2x)
Gel Extraction of PCR 6
µl | type |
---|---|
34.8 | H2O |
13.0 | Taq Buffer |
13.0 | dNTPs |
1.3 | oIG2002 |
1.3 | oIG2005 |
1.6 | Taq Polymerase |
5.0 | H2O for each reaction, containing picked colony |
- Annealing: 60°C
- Extension: 68°C
- Extension: 2 min 10 sec.
- Cycles: 30
Results of Colony PCR: Clones 5, 6 and 7 were striked out. |
name | ng/µl |
---|---|
pIG2004, No. 5 | 207.9 |
pIG2004, No. 6 | 180.0 |
pIG2004, No. 7 | 198.1 |
Clones were sent to sequencing at GATC
oIG0007 sequencing reveals that the KRAB fragment was succesfully fused to Cas9 in each case. Further sequecing will be done to test for correct N-terminal assembly and H840A mutation. |
µl | type |
---|---|
10 | Q5-HF Reaction Buffer |
1 | pX334 |
1 | oIG2002 |
1 | oIG2007 |
2.5 | dNTPs |
1.5 | DMSO |
0.5 | Q5-HF Polymerase |
Add to 50 | H2O |
- Annealing: 60°C
- Extension: 2 min 40 sec.
- Cycles: 18
Agarose Gelelectrophoreses of PCR fragments No. 6
f.l.t.r.: marker, PCR 6 (3908 bp), empty, PCR 6 (2x) |
Gel Extraction of PCR 6
name | ng/µl |
---|---|
Fragment No. 6 | 43.6 |
Gibson Assembly of pIG2005 (dCas9)
- Preparation of 5µl DNA mix, containing all four fragments
- Melting a 15µl master-mix on ice, until it is ready to use
- Addition of 5µl of DNA mix to the master mix
- Immediately put the mix on 50°C and incubate for 1h
- 3 min on RT
- 3 min on ice
- Transformation of 4 µl to TOP10 chemically competent E-coli cells
Preparation scheme for Gibson Assembly fragment mix |
24. May
Digest of pIG2004 (No. 5, 6 and 7) with BbsI
µl | type |
---|---|
Volume | DNA |
16 | pIG2004 |
10 | NEB-Buffer 2.1 |
2 | BbsI |
72 | H2O |
- Temp.: 37°C
- Incubation time: 2h
Agarose Gelelectrophoreses of pIG2004 Digests
f.l.t.r.: marker, pX334, empty, digested pIG2004 5 (2x), 6 (2x) and 7 (2x). Upper bands of clone no. 5 and 6 digests were cut out for Gel Extraction. |
Gel Extraction of pIG2004 digest
name | ng/µl |
---|---|
pIG2004 (5) | 38.0 |
pIG2004 (6) | 17.0 |
Evaluation of Gibson Assembly of 23. May
- Colony growth
- Five clones were striked out
27. May
Evaluation of pIG2004 sequencing results (No. 5 and 6)
- H840A mutation was inserted in both cases, leading to catalytic inactivation of Cas9
- a 300 bp gap was inserted within the CAG promoter, probably due to error-prone PCR amplification
Very GC-rich part on pIG2004 template (part of the CAG promoter lacks nearly 300 bp after having been amplified via PCR and subsequently Gibson-assembled. |
June
04. June
Minipreps of pIG2005 clones striked out on 23. May
name | ng/µl |
---|---|
pIG2005 - No. 1 | 77.1 |
pIG2005 - No. 2 | 68.4 |
pIG2005 - No. 3 | 59.6 |
pIG2005 - No. 4 | 43.7 |
pIG2005 - No. 5 | 61.2 |
07. June
Fixing of the 300 bp CAG promoter gap for Cas9-KRAB
Restriction digest of pIG2004 and pIG2005 - 1
µl
type
8
pIG2004 - No. 5 or pIG2005 - No. 1
8
NEB-Buffer 4
2
AgeI-HF
2
NotI-HF
2
SacII
2
BSA
Add to 80µl
H2O
Restriction digest of pX334 - 2
µl
type
8
pX334 DNA
8
NEB-Buffer 4
2
KpnI-HF
2
NotI-HF
2
SacI-HF
2
BSA
Add to 80µl
H2O
Restriction digest of pX334 - 3
µl
type
8
pX334 DNA
8
NEB-Buffer 4
2
KpnI-HF
2
AgeI-HF
2
BSA
Add to 80µl
H2O
- Temp.: 37°C
- Incubation time: 1h
- Expected fragment lengths: 1 - 4500 bp (Cas9-KRAB), 2 - 4900 bp (Backbone), 3 - 890 bp (CAG promoter)
Top left. Upper band: Cas9-KRAB fragment, Top right. Upper band: dCas9 fragment. Bottom left. Upper band: pX334 Backbone fragment.
Top right. Lowest band (blurred): CAG promoter fragment.
Gel Extraction of digest fragments
µl | type |
---|---|
8 | pIG2004 - No. 5 or pIG2005 - No. 1 |
8 | NEB-Buffer 4 |
2 | AgeI-HF |
2 | NotI-HF |
2 | SacII |
2 | BSA |
Add to 80µl | H2O |
µl | type |
---|---|
8 | pX334 DNA |
8 | NEB-Buffer 4 |
2 | KpnI-HF |
2 | NotI-HF |
2 | SacI-HF |
2 | BSA |
Add to 80µl | H2O |
Restriction digest of pX334 - 3
µl
type
8
pX334 DNA
8
NEB-Buffer 4
2
KpnI-HF
2
AgeI-HF
2
BSA
Add to 80µl
H2O
- Temp.: 37°C
- Incubation time: 1h
- Expected fragment lengths: 1 - 4500 bp (Cas9-KRAB), 2 - 4900 bp (Backbone), 3 - 890 bp (CAG promoter)
Top left. Upper band: Cas9-KRAB fragment, Top right. Upper band: dCas9 fragment. Bottom left. Upper band: pX334 Backbone fragment.
Top right. Lowest band (blurred): CAG promoter fragment.
Gel Extraction of digest fragments
µl | type |
---|---|
8 | pX334 DNA |
8 | NEB-Buffer 4 |
2 | KpnI-HF |
2 | AgeI-HF |
2 | BSA |
Add to 80µl | H2O |
- Temp.: 37°C
- Incubation time: 1h
- Expected fragment lengths: 1 - 4500 bp (Cas9-KRAB), 2 - 4900 bp (Backbone), 3 - 890 bp (CAG promoter)
Top left. Upper band: Cas9-KRAB fragment, Top right. Upper band: dCas9 fragment. Bottom left. Upper band: pX334 Backbone fragment. |
Top right. Lowest band (blurred): CAG promoter fragment. |
name | ng/µl |
---|---|
Cas9-KRAB | 26.7 |
dCas9 | 5.0 |
pX334 Backbone | 14.4 |
CAG promoter | 5.0 |
Ligation and transformation of intact pIG2004 and pIG2005
Ligation of pIG2005
µl
type
11.2
CAG fragment
4.4
dCas9 fragment
1.4
pX334 backbone
2
T4 ligase buffer
1
T4 ligase
Ligation of pIG2004
µl
type
0.8
Cas9-KRAB fragment
14.8
CAG fragment
1.4
pX334 backbone
2
T4 ligase buffer
1
T4 ligase
08. June
Evaluation of Transformations of pIG2005 and pIG2004
- pIG2004 (30 µl strikeout): 24 colonies
- pIG2005 (30 µl strikeout): 15 colonies
- 12 colonies of both approaches were striked out for further sequencing
09. June
Minipreps of pIG2004 and pIG2005
name
ng/µl
pIG2004 - No. 1
198.4
pIG2004 - No. 2
203.6
pIG2004 - No. 3
202.0
pIG2004 - No. 4
187.3
pIG2004 - No. 5
195.3
pIG2004 - No. 6
211.2
pIG2004 - No. 7
187.4
pIG2004 - No. 8
201.1
pIG2004 - No. 9
201.7
pIG2004 - No. 10
206.3
pIG2004 - No. 11
212.3
pIG2004 - No. 12
174.6
pIG2005 - No. 1
202.0
pIG2005 - No. 2
195.3
pIG2005 - No. 3
189.9
pIG2005 - No. 4
221.4
pIG2005 - No. 5
205.4
pIG2005 - No. 6
194.4
pIG2005 - No. 7
206.7
pIG2005 - No. 8
206.3
pIG2005 - No. 9
220.4
pIG2005 - No. 10
192.9
pIG2005 - No. 11
188.8
pIG2005 - No. 12
192.1
- Colonies No. 2 and 3 of pIG2004 were sent to sequencing, intending to validate (i) KRAB-insertion (ii) H840A conversion and (iii) an intact CAG promoter
- Colonies No. 1 and 4 of pIG2005 were sent to sequencing, intending to validate (i) H840A conversion and (ii) an intact CAG promoter
11. June
Evaluation of Sequencing results of pIG2004 and pIG2005
- pIG2004 clone No. 2 misses the KRAB fragment, but contains the CAG promoter
- pIG2004 clone No. 3 contains KRAB fragment, H840A and the CAG promoter - and will therefore be used for further studies
- pIG2005 clones No. 1 and 4 contain both H840 mutation and the CAG promoter
Restriction digest of pIG2004 and pIG2005 with BbsI
µl
type
6
DNA
4
NEB-Buffer 2.1
2
BbsI
Add to 40µl
H2O
F.l.t.r.: marker, pIG2004 undigested, pIG2004 digested, pIG2005 undigested, pIG2005 digested (2x)
Gel Extraction of digested vectors pIG2004 and pIG2005
µl | type |
---|---|
11.2 | CAG fragment |
4.4 | dCas9 fragment |
1.4 | pX334 backbone |
2 | T4 ligase buffer |
1 | T4 ligase |
µl | type |
---|---|
0.8 | Cas9-KRAB fragment |
14.8 | CAG fragment |
1.4 | pX334 backbone |
2 | T4 ligase buffer |
1 | T4 ligase |
name | ng/µl |
---|---|
pIG2004 - No. 1 | 198.4 |
pIG2004 - No. 2 | 203.6 |
pIG2004 - No. 3 | 202.0 |
pIG2004 - No. 4 | 187.3 |
pIG2004 - No. 5 | 195.3 |
pIG2004 - No. 6 | 211.2 |
pIG2004 - No. 7 | 187.4 |
pIG2004 - No. 8 | 201.1 |
pIG2004 - No. 9 | 201.7 |
pIG2004 - No. 10 | 206.3 |
pIG2004 - No. 11 | 212.3 |
pIG2004 - No. 12 | 174.6 |
pIG2005 - No. 1 | 202.0 | pIG2005 - No. 2 | 195.3 | pIG2005 - No. 3 | 189.9 | pIG2005 - No. 4 | 221.4 | pIG2005 - No. 5 | 205.4 | pIG2005 - No. 6 | 194.4 | pIG2005 - No. 7 | 206.7 | pIG2005 - No. 8 | 206.3 | pIG2005 - No. 9 | 220.4 | pIG2005 - No. 10 | 192.9 | pIG2005 - No. 11 | 188.8 | pIG2005 - No. 12 | 192.1 |
µl | type |
---|---|
6 | DNA |
4 | NEB-Buffer 2.1 |
2 | BbsI |
Add to 40µl | H2O |
F.l.t.r.: marker, pIG2004 undigested, pIG2004 digested, pIG2005 undigested, pIG2005 digested (2x) |
Gel Extraction of digested vectors pIG2004 and pIG2005
name | ng/µl |
---|---|
pIG2004 | 16.1 |
pIG2005 | 18.8 |
12. June
Oligo annealing of target crRNAs
µl | type |
---|---|
3 | Oligo No. 1 |
3 | Oligo No. 2 |
69 | NEB-Buffer 2 |
- Samples were heated up to 95 degrees for 5 minutes. Gradient cool down to room temperature for 2 hours.
- Pair 1: oIG2010/oIG2011 - EMX1
- Pair 2: oIG2022/oIG2023 - Target 200 bp upstream of SEAP transcription initiation on pKM006
- Pair 3: oIG6008/oIG6009 - CMV
13-20. June
Ligation and transformation of crRNAs with Cas9-KRAB and dCas9
Ligation of pIG2013
µl | type |
---|---|
15 | Annealed EMX1 |
3 | oIG2004 |
2 | T4 ligase buffer |
1 | T4 ligase |
Ligation of pIG2017
µl | type |
---|---|
15 | Annealed EMX1 |
3 | oIG2005 |
2 | T4 ligase buffer |
1 | T4 ligase |
Ligation of pIG2019
µl | type |
---|---|
15 | Annealed SEAP target |
3 | oIG2004 |
2 | T4 ligase buffer |
1 | T4 ligase |
Ligation of pIG2020
µl | type |
---|---|
15 | Annealed SEAP target |
3 | oIG2005 |
2 | T4 ligase buffer |
1 | T4 ligase |
Ligation of pIG2011
µl | type |
---|---|
15 | Annealed CMV target |
3 | oIG2004 |
2 | T4 ligase buffer |
1 | T4 ligase |
Ligation of pIG2015
µl | type |
---|---|
15 | Annealed CMV target |
3 | oIG2005 |
2 | T4 ligase buffer |
1 | T4 ligase |
Evaluation of transformation results
- Except for pIG2020, colonies were grown in each case
- 6 colonies of pIG2011, pIG2013, pIG2015, pIG2017 and pIG2019 were respectively striked out
- All attempts had to be repeated at least twice, as BbsI digestion often lead to unspecific digestion of direct-repeats next to crRNA insertion spacer.
18-25. June
MIDI-preps of pIG2004, pIG2005, pIG2011, pIG2013, pIG2015, pIG2017, pIG2019 and also pIG9000
name | ng/µl |
---|---|
pIG2004 (Cas9-KRAB) | 400 |
pIG2005 (dCas9) | 203 |
pIG2011 (Cas9-KRAB vs. CMV) | 610 |
pIG2013 (Cas9-KRAB vs. EMX1) | 609 |
pIG2015 (dCas9 vs. CMV) | 460 |
pIG2017 (dCas9 vs. EMX1) | 520 |
pIG2017 (dCas9 vs. EMX1) | 520 |
pIG2019 (Cas9-KRAB vs. SEAP) | 430 |
pIG9000 (Cas9 vs. EMX1) | 460 |
- MIDIs had to be repeated three times
- During the first attempt, an unkown white precipitate was obtained - which could not be separated from the desired DNA.
- Within the second attempt, DNA was not adequately treated during isopropanol treatment (has to be shaken).
25.-27. June
Test of Cas9-KRAB on Western Blots
- Cas9-KRAB and dCas9, both targetting the CMV promoter were respectively co-transfected to HEK 293 cells with another plasmid, encoding CMV driven CNK.
- Both attempts were performed repeatedly, and cell lyses were than to be performed 12, 18, 36 and 42 hours post-transfection.
- Transfections were performed with PEI
- Lyses were performed with modified RIPA buffer
- Biological double attempts were respectively done
Transfection schemes for one 6-well plate
Cas9-KRAB vs. CMV of CNK (2x)
name | µg |
---|---|
CNK | 6 |
pIG2011 | 12 |
dCas9 vs. CMV of CNK (2x)
name | µg |
---|---|
CNK | 6 |
pIG2015 | 12 |
CNK negative control (2x)
name | µg |
---|---|
CNK | 6 |
Cas9-KRAB and dCas9 expression controls on one well
name | µg |
---|---|
pIG2004 | 1 |
pIG2005 | 1 |
Results
??? |
??? |
- We expected a stronger decrease in CNK levels, when combining it with Cas9-KRAB than with dCas9
- Cas9-KRAB was able to highly efficiently repress CMV driven CNK expression
- Cas9-KRAB was expressed.
29. June
Test of Cas9-KRAB vs. CMV-driven GFP
- Transfection of pIG6000 (CMV::GFP) in combination with either pIG2011 (Cas9-KRAB + crRNA agaisnt CMV) or Mock DNA
- Cells were seeded previously seeded on 6-well plates
- Incubation for about 36 hours
dCas9 vs. CMV of GFP
name | µg |
---|---|
CMV-GFP | 1 |
pIG2011 (Cas9-KRAB) | 2 |
PEI | 9 |
Transfection scheme |
July
01. July
Fluorescence Microscopy Evaluation
Fluorescence Images |
ImageJ: Cells treated with GFP and with pRSET (Mock-DNA)
Cell | Size | Intensity |
---|---|---|
1 | 208 | 2920.9 |
2 | 146 | 3945.0 |
3 | 238 | 3064.8 |
4 | 166 | 3699.3 |
5 | 238 | 2071.3 |
6 | 192 | 2219.1 |
7 | 172 | 3819.9 |
8 | 228 | 3235.1 |
9 | 247 | 3510.0 |
10 | 146 | 3213.6 |
11 | 228 | 1977.4 |
12 | 224 | 3436.5 |
13 | 224 | 3436.5 |
14 | 136 | 1872.9 |
15 | 140 | 1611.4 |
ImageJ: Cells treated with GFP and Cas9-KRAB-DNA
Cell | Size | Intensity |
---|---|---|
1 | 268 | 2407.6 |
2 | 176 | 3329.2 |
3 | 217 | 1336.1 |
4 | 166 | 1138.5 |
5 | 102 | 2844.8 |
6 | 217 | 936.6 |
7 | 148 | 1236.4 |
8 | 214 | 1032.9 |
9 | 177 | 1566.6 |
10 | 96 | 1461.9 |
11 | 228 | 1977.4 |
12 | 938 | 3371.7 |
13 | 102 | 992.9 |
14 | 210 | 982.1 |
15 | 112 | 1016.6 |
- Mean of GFP fluorescence intensity of cells treated with Cas9-KRAB is 1409.4 (SD: 880.6), cells co-transfected with Mock-DNA revealed 2718 (SD: 786.6)
- Results should be of low validity, but are potentially indicative towards a repressive effect of Cas9-KRAB
02. July
MIDI-prep of pIG2005, pIG2019 and pRSET (Mock-DNA)
Failed in each case, for unknown reasons (white salt fall-out after elution from column). Attempt will be repeated on the next day.
03. July
MIDI-prep of pIG2005, pIG2019 and pRSET (Mock-DNA)
name | ng/µl |
---|---|
pIG2005 (dCas9) | 470 |
pIG2019 (Cas9-KRAB vs. SEAP target) | 430 |
pRSET (Mock DNA) | 790 |
04. July
Transfection of pIG2019 in combination with pKM006 (SEAP plasmid) and pSAM200 (SV40::tetR-VP16)
- Biological tetraplicates (each attempt was repeated independently, for four different wells of one 6-well plate
- Negative control: pKM006 (500 ng) + Mock-DNA (2500 ng)
- Positive control: pKM006 (500 ng) + pSAM200 (500 ng) + Mock-DNA (2000 ng)
- Cas9-KRAB test: pKM006 (500 ng) + pSAM200 (500 ng) + pIG2019 (2000 ng)
07. July
SEAP measurement
- 300µl of medium supernatants were removed from each well
- Samples were heated to 65°C for 30 minutes
- Centrifugation at 600 rpm (table top) for 1 minute
- 80µl of each supernatant was transfered to one well of a 96-well plate, technical triplicates for each sample were pipetted
- 100µl 2x SEAP Buffer was added to each well
- 20µl pNPP substrate was added to each well
- Air bubbles were quickly removed
- Measurement was taken with a plate reader, for two consecutive hours with an interval lenght of one minute. Detection wavelength was 405 nm
SEAP Results |
September
01. September
Test of Cas9-KRAB vs. CMV-driven GFP, Repetition of the Experiment from 29. June on 24 well plates
dCas9 vs. CMV of GFP
name | µg |
---|---|
CMV-GFP | 0,04 |
pIG2011 (Cas9-KRAB) | 0,46 |
PEI | 1,5 |
dCas9 vs. CMV of GFP
name | µg |
---|---|
CMV-GFP | 0,04 |
pRSET (Mock-DNA) | 0,46 |
PEI | 1,5 |
03. September
Fluorescence Microscopy of KRAB-mediated GFP-repression
Results of GFP repression. Fluorescence images were taken with a light exposure time of 250 ms (80% saturation). Experiment shall be repeated with off-target controls and parallel Flow Cytometry analysis. |
09. September
Retrafo of Plasmids for the Midiprep
-
Following plasmids are needed for activation and repression repeats:
- pRSet
- pKM600
- pKM604
- pKM605
- pKM606
- pKM607
- pKM603
- pIG2017
- pIG2013
- pSAM200
10. September
Midiprep
Plasmids were prepped with the Midiprep kit of Promega according to the manufacturer's protocol.
Seeding of cells
4 24-well plates were seeded with 65,000 cells per well
11. September
Transfection
- 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
- 0.75 µg of the DNA of interest were prepaired in another Eppi .
- Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
- Solution was spread drop-wise to the cells in the dish
Medium change
Medium was changed after 5 h.13. September
Preparation for analyses
- Supernatant was collected for SEAP measurement.
- Cells were frozen at - 80 °C for later on Western blot and luminescence measurement.
14. September
SEAP measurement
- Collected supernatant and fresh cell culture medium was heated at 65 °C for 30 min, then centrifuged at 1250 g for 1 min.
- Supernatant was diluted in cell culture medium 1:4 (25 µl supernatant + 75 µl medium).
- 80 µl of diluted supernatant was given to 100 µl of SEAP buffer.
- After addition of 20 µl pNPP (substrate of SEAP) the absorbance of the mix was measured every minute for 2 h at 405 nm.
16. September
Cell lysis
- 250 µl of lysis buffer (containing Tris-HCl, EGTA, MgSO4, DTT and protease inhibitor) were applied to the thawn cells of each well.
- Incubation on ice for 10 min.
- Centrifugation at 15,000 g for 4 min.
Renilla luminescence measurement
- 80 µl of supernatant were pipetted in a white 96 well plate.
- Measurement of luminescence (every 2 min for 30 min) immediately after addition of 20 µl Renilla substrate in PBS.
SEAP activity normalized to Renilla expression |
Red bars: When targeting the same locus, Cas9 leaded to less SEAP expression in combination with Cas9-VP16 than Cas9-KRAB in the same combination.
17. September
Protein precipitation
As the cell lysis needed to be done with a high amount of lysis buffer, proteins are too much diluted for western blotting. Thus, they were precipitated:
- The remaining lysates of the triplikates (~ 80 µl) were put together in a new epi.
- Addition of TCA to a final concentration of 10 % and 1 µg BSA.
- Incubation for 30 min on ice.
- Centrifugation for 30 min at full speed and 4 °C.
- Removal of supernatant and addition of 500 µl acetone.
- Incubation on ice over night.
- Centrifugation for 15 min at full speed and 4 °C.
- Removal of acetone and nair drying for 20 min.
- Addition of 20 µl 2x SDS-loading dye and 0.5 µl Tris-HCl (pH = 8.8).
- Heating at 95 °C for 5 min.
18. September
SDS-gel run
SDS-gel was loaded with 18 µl of each sample. A voltage of 80 V was applied till the loading dye bands reached the border between collection and separation gel, then the voltage was increased to 120 V.
Western Blotting
- Activation of PVDF-membrane for 10 min in methanol.
- Short incubation of Whatman papers and the PVDF-membrane in transfer buffer.
- Loading of western blot apperature: (-) - Whatman paper - SDS-gel - PVDF-membrane - Whatman paper - (+).
- 200 mA were applied for 1.5 h.
Antibody treatment I
- Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 1 h.
- Incubation with anti HA antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.
- 3 x washing with TBS-T for 5 min, each.
- Incubation with anti mouse antibody for 1 h.
- 4 x washing with TBS-T for 10 min, each.
- Measurement of luminescence after addition of ECL I + ECL II solution.
- Air drying of the membrane for further antibody treatments.
19. September
Seeding of cells
3 24-well plates were seeded with 65,000 cells per well.
Antibody treatment II
- Reactivation in methanol.
- 1 x washing with TBS-T for 5 min.
- Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 30 min.
- Incubation with anti beta-actin antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.
- 3 x washing with TBS-T for 5 min, each.
- Incubation with anti mouse antibody for 1 h.
- 4 x washing with TBS-T for 10 min, each.
- Measurement of luminescence after addition of ECL I + ECL II solution (500 µl each).
Western blot |
20. September
Transfection
- 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
- 0.75 µg of the DNA of interest were prepaired in another Eppi .
- Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
- Solution was spread drop-wise to the cells in the dish
Ratio of DNA amount (mass):
reporter plasmid (SEAP) : effector plasmid (Cas9) : RNA plasmid
1 : 4 : 4
Medium change
Medium was changed after 4.5 h.22. September
Medium removal
- Supernatant was collected for SEAP measurement.
- Cells were frozen at - 80 °C for later on Western blotting.
Preparation for SEAP measurement
Collected supernatant and fresh cell culture medium was heated at 65 °C for 30 min, then centrifuged at 1250 g for 1 min.
23. September
SEAP measurement
- Supernatant of wells containing CMV:SEAP was diluted in cell culture medium 1:10 (10 µl supernatant + 90 µl medium).
- 80 µl of (diluted) supernatant was given to 100 µl of SEAP buffer.
- After addition of 20 µl pNPP (substrate of SEAP) the absorbance of the mix was measured every minute for 2 h at 405 nm.
SEAP activity [U/L] pIG2004: Cas9-KRAB (not in iGEM standard); pIG2005: Cas9 (not in iGEM standard); PhyB: negative control; all other Cas9 constucts are in iGEM standard (with the indicated promoter) |
Seeding of cells
4 24-well plates were seeded with 65,000 cells per well
24. September
Repeat of seeding of cells
4 24-well plates were seeded with 65,000 cells per well again, as the cell density of the first seeding was too low.
25. September
Transfection
- 40 µl Opti-MEM + 0.75 µg of the DNA of interest were mixed in a 1.5 ml Eppi.
- Addition of 2.25 µl PEI-solution, vortexing for 10 s and incubation for at least 15 min at RT
- Solution was spread drop-wise to the cells in the dish
Ratio of DNA amount (mass):
reporter plasmid (SEAP) : effector plasmid (Cas9) : RNA plasmid
1 : 4 : 4
Additionally a plasmid coding for Renilla luciferase was cotransfected (5 % of DNA amount)
26. September
Medium change
Medium was changed after 8 h.Cell lysis of transfection of 20. September
- 80 µl of RIPA lysis buffer were applied to the thawn cells of each well.
- Incubation on ice for 10 min.
- Sonification 3 x 30 s at maximum.
- Centrifugation at 10,000 g for 10 min.
- 40 µl of supernatant were mixed with 10 µl 5 x SDS sample buffer.
- Heating for 5 min at 95 °C.
SDS-gel run
SDS-gel was loaded with 40 µl of each sample. A voltage of 80 V was applied till the loading dye bands reached the border between collection and separation gel, then the voltage was increased to 120 V.
Western Blotting
- Activation of PVDF-membrane for 10 min in methanol.
- Short incubation of Whatman papers and the PVDF-membrane in transfer buffer.
- Loading of western blot apperature: (-) - Whatman paper - SDS-gel - PVDF-membrane - Whatman paper - (+).
- 200 mA were applied for 1.5 h.
Antibody treatment I
- Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 1 h.
- Incubation with anti HA antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.
- 3 x washing with TBS-T for 5 min, each.
- Incubation with anti mouse antibody for 1 h.
- 4 x washing with TBS-T for 10 min, each.
- Measurement of luminescence after addition of ECL I + ECL II solution.
- Air drying of the membrane for further antibody treatments.
Western blot of the SEAP assay from 23.09. CAG:dCas was strongly expressed, whereas CMV:dCas and dCas-KRAB were not visible on the western blot. Nevertheless a effect has been detected with the SEAP assay, so there should be an expression, but maybe too low for detection. |
27.09.13
Medium removal
- Supernatant was collected for SEAP measurement 48 h after transfection.
- Cells were frozen at - 80 °C for later on Western blotting.
28.09.13
SEAP measurement
- Collected supernatant and fresh cell culture medium was heated at 65 °C for 30 min, then centrifuged at 1250 g for 1 min.
- Supernatant of wells containing CMV:SEAP was diluted in cell culture medium 1:15 (6.7 µl supernatant + 93.3 µl medium).
- 80 µl of (diluted) supernatant was given to 100 µl of SEAP buffer.
- After addition of 20 µl pNPP (substrate of SEAP) the absorbance of the mix was measured every minute for 2 h at 405 nm.
Cell lysis for lyciferase measurement and western blot
- 250 µl of lysis buffer (containing Tris-HCl, EGTA, MgSO4, DTT and protease inhibitor) were applied to the thawn cells of each well.
- Incubation on ice for at least 10 min.
- Centrifugation at 2,200 g for 30 min.
Luciferase luminescence measurement
- 80 µl of supernatant were pipetted in a white 96 well plate.
- Measurement of luminescence (every 2 min for 15 min) immediately after addition of 20 µl Renilla substrate in PBS.
SEAP-Assay: Results All different tested loci showed no clear result. dCas9-KRAB under the control of the CMV promotor showed no significant repression in comparison to the off-target and CRISPRi control. dCas9-KRAB under the control of the SV40 promotor showed no repression at all. Maybe targeting a gene coded on a plasmid is a problem for dCas9-KRAB to repress. |
29.09.13
Protein precipitation
As the cell lysis needed to be done with a high amount of lysis buffer, proteins are too much diluted for western blotting. Thus, they were precipitated:
- 340 µl of acetone was added to 85 µl of the remaining lysates of one well per triplicate.
- Incubation at - 20 °C for 1:15 h.
- Centrifugation for 5 min at 10,000 g and 4 °C.
- Removal of acetone and air drying for 5 min.
- Addition of 25 µl 2x SDS-loading dye.
- Heating at 95 °C for 5 min.
SDS-gel run
SDS-gel was loaded with 20 µl of each sample. A voltage of 80 V was applied untill the loading dye bands reached the border between collection and separation gel, then the voltage was increased to 120 V.
Western Blotting
- Activation of PVDF-membrane for 10 min in methanol.
- Short incubation of Whatman papers and the PVDF-membrane in transfer buffer.
- Loading of western blot apperature: (-) - Whatman paper - SDS-gel - PVDF-membrane - Whatman paper - (+).
- 200 mA were applied for 1.5 h.
Antibody treatment I
- Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 1 h.
- Incubation with anti HA antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.
- 3 x washing with TBS-T for 5 min, each.
- Incubation with anti mouse antibody for 1 h.
- 4 x washing with TBS-T for 10 min, each.
- Measurement of luminescence after addition of ECL I + ECL II solution.
- Air drying of the membrane for further antibody treatments.
Western blot of the SEAP assay from 28.09. Western blotting did not work well. Maybe lysis due to relative long incubation time on Renilla lysis buffer destroyed cells to much beforehand. So maybe only degradation products are visible. |