Team:Freiburg/Notebook/lab multiple targeting

From 2013.igem.org

(Difference between revisions)
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→ pIG7003a, heat shock transfection in E. coli (according to protocol)<br>
→ pIG7003a, heat shock transfection in E. coli (according to protocol)<br>
→ pIG7004a, heat shock transfection in E. coli (according to protocol)<br> </p>
→ pIG7004a, heat shock transfection in E. coli (according to protocol)<br> </p>
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</div>
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<div id="tag">
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  <h2> 31.05.13 </h2>
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<h3> Digest of pIG7001a </h3>
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<p> used: colonie 1 (pIG7001a), NcoI, PstI HF, XbaI </p>
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<div id="floatleft">
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<table class="tabelle">
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<tr>
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<th> &micro;l </th>
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<th> type </th>
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</tr>
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<tr>
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<td> 5 </td>
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<td> DNA </td>
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</tr>
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<tr>
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<td> 1 </td>
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<td> NEB-Buffer 4 </td>
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</tr>
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<tr>
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<td> 0,5 </td>
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<td> XbaI </td>
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</tr>
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<tr>
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<td> 0,1 </td>
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<td> BSA </td>
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</tr>
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<tr>
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<td> Add to 10&micro;l </td>
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<td> H<sub>2</sub>O </td>
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</tr>
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</table>
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</div>
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<div id="floatleft">
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<table class="tabelle">
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<tr>
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<th> &micro;l </th>
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<th> type </th>
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</tr>
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<tr>
 +
<td> 5 </td>
 +
<td> DNA </td>
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</tr>
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<tr>
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<td> 1 </td>
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<td> NEB-Buffer 4 </td>
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</tr>
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<tr>
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<td> 0,5 </td>
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<td> NcoI HF </td>
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</tr>
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<tr>
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<td> Add to 10&micro;l </td>
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<td> H<sub>2</sub>O </td>
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</tr>
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</table>
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</div>
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<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 5 </td>
 +
<td> DNA </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> NEB-Buffer 4 </td>
 +
</tr>
 +
<tr>
 +
<td> 0,5 </td>
 +
<td> PstI HF </td>
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</tr>
 +
<tr>
 +
<td> Add to 10&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
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</tr>
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</table>
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</div>
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<div id="floatright">
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<ul>
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<li> Temp.: 37&deg;C </li>
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<li> Incubation time: 1h </li>
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</ul>
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</div>
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<table id="floatleft">
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<tr>
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<td> <img src="https://static.igem.org/mediawiki/2013/9/95/Freiburg_2013_projekt7_05-31_Digest_%28Geneious%29.png"> </td>
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</tr>
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<tr>
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<td> expected bands </td>
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</tr>
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</table>
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<table class="gelpic">
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<tr>
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<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/7/70/Freiburg_2013_projekt7_05-31_3x_digest.Jpg"> </td>
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</tr>
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<tr>
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<td> pIG7001a digest <br>
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left to right:<br>
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1: ladder (1kb)<br>
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2: XbaI <br>
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3: NcoI <br>
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4: PstI <br>
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5: negative control </td>
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</tr>
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</table>
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<p> XbaI did only cut once instead of twice, NcoI and PstI cut as expected </p>
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<h3> Sequencing of pIG7001a(1) </h3>
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<p> GATC-Watch-box: 706894 <br>
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→ Sequence of pIG7001a ok </p>
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 +
<div id="june">
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<p id="h2">
 +
June
 +
</p>
</div>
</div>

Revision as of 13:15, 24 September 2013

crRNA - Multiple targeting

May

28.05.13

PCR 1

for Gibson Assembly

Fragment 1 pACGFP1-Golgi oIG7001-F oIG7001-R 2209 bp
Fragment 2 pACGFP1-Golgi oIG7002-F oIG7002-R 2126 bp
Fragment 3 pKM006 oIG7003-F oIG7003-R 1100 bp
Stuff Volume
Template(Plasmid) 0,5µl
Primer(10 µM) each 1µl
Q5 Polymerase 0,5µl
dNTPs(2,5mM) 2µl
Q5 Buffer(5x) 4µl
Water 11µl
total 20µl

Program

98°C 5min Denaturation
98°C 30s Denaturation
60°C 30s Annealing
72°C 35s/kb Elongation
72°C 10min final Elongation
4°C hold

14 cycles

15µl of each product from PCR 1 were load on gel:

Products PCR 1
left to right:
1: ladder (1kb)
2: Fragment 1 (2209bp)
3:Fragment 2 (2126bp)
4:Fragment 3 (1100bp)

PCR 2

for Gibson Assembly
template: products from PCR 1

Stuff Volume
Template(PCR product) 2µl
Primer(10 µM) each 1µl
Q5 Polymerase 1µl
dNTPs(2,5mM) 5µl
Q5 Buffer(5x) 10µl
Water 30µl
total 50µl

Program

98°C 5min Denaturation
98°C 30s Denaturation
60°C 30s Annealing
72°C 35s/kb Elongation
72°C 10min final Elongation
4°C hold

18 cycles

Concentration of PCR-Products:
1: 990 ng/µl 2: 900 ng/µl 3: 870 ng/µl

4µl of each product from PCR 2 were load on gel:

Products PCR 2
left to right:
2: ladder (1kb)
3: Fragment 1 (2209bp))
4: Fragment 2 (2126bp)
5: Fragment 3 (1100bp)

Gibson Assembly

Calculation of DNA mix for Gibson Assembly:
concentration [ng/µl] * size [kbp] * 0.00023 [1/(ng*kb)]
1h in 1 aliquot of Gibson mix (according to protocol)
→ pIG7001a , heat shock transfection in E. coli (according to protocol)

29.05.13

6 colonys pIG7001a were picked and plated

Colony PCR

from 6 colonies with pIG7001a (Gibson Assembly 28-05)

Product pKM006 oIG7003-F oIG7003-R 1100bp
Volume Stuff
Template (Bacteria from colonie)
1µl Taq Standart buffer (10x)
0,5µl Primer1
0,5µl Primer2
2.5µl dNTPs
0.5µl Taq polymerase
7µl H2O
10µl total

Program

98°C 7min Denaturation
98°C 30s Denaturation
67°C 30s Annealing
72°C 50s Elongation
72°C 10min final Elongation
4°C hold

30 cycles

10µl of each PCR product were load on a gel:

Products Colony-PCR
left to right:
1: ladder (1kb)
2-7: colonie 1-6 from pIG7001a (gibson assembly 05-28)
8: negativ control
9: positive control (template is pKM006)
PCR did not work

30.05.13

6 minipreps of pIG7001a:
cancentrations: 196 ng/µl, 175 ng/µl, 195 ng/µl, 133 ng/µl, 203 ng/µl, 197 ng/µl

Double-Digest of pIG7001a

used: minipreps of colonies 1-6, NgoMIV, PstI HF

µl type
5 DNA
1 NEB-Buffer 4
0.5 Pst1 HF
0.5 NgoMIV
Add to 10 µl H2O
  • Temp.: 37°C
  • Incubation time: 1h

the whole mix was load on a gel:

expected bands
Products double digest:
pIG7001a (Prep 1-6) with Pst1 and NgoMIV

PCR 1

colonie 1 was used (oIG7001a)
for Gibson Assembly 2:

Fragment 1 pIG7001a oIG7004-F oIG7004-R 2274 bp
Fragment 2 pIG7001a oIG7005-F oIG7005-R 2248 bp
Fragment 3 GW1-Peredox_mCherry-NLS oIG7006-F oIG7006-R 741 bp
for Gibson Assembly 3:
Fragment 1 pIG7001a oIG7004-F oIG7004-R 2274 bp
Fragment 2 pIG7001a oIG7005-F oIG7005-R 2248 bp
Fragment 3 pFucci-G1-Orange oIG7007-F oIG7007-R 659 bp
for Gibson Assembly 4:
Fragment 1 pIG7001a oIG7004-F oIG7004-R 2274 bp
Fragment 2 pIG7001a oIG7005-F oIG7005-R 2248 bp
Fragment 3 pDS47-BFP-dGEM-trunc oIG7008-F oIG7008-R 679 bp
µl type
4 Q5-HF Reaction Buffer
0,5 Template
1 each Primer
5 dNTPs
0.5 Q5-HF Polymerase
Add to 20 H2O
  • Gibson 1 program was used

5µl of each product were load on a gel

Products of PCR 1

Product 3 from Gibson 2 is missing → 0,2 µl extra plasmid template was added for PCR 2

PCR 2

for gibson assembly 2, 3, and 4
template: products from 1. PCR

µl type
4 Q5-HF Reaction Buffer
2 Template
1 each Primer
5 dNTPs
0.5 Q5-HF Polymerase
Add to 20 H2O
  • Gibson 2 program was used

concetration of PCR products:
Gibson 2: 1415 ng/µl, 1417 ng/µl, 1315 ng/µl
Gibson 3: 2874 ng/µl, 1436 ng/µl, 337 ng/µl
Gibson 4: 324 ng/µl, 325 ng/µl, 2047 ng/µl

5µl of each PCR product were load on a gel

Products of PCR 2

Gibson Assembly

volumes were calculated with excel sheet with concentration and length:
used volumes:
Gibson 2: 0,3 µl, 0,3 µl, 0,1 µl
Gibson 3: 0,15 µl, 0,3 µl, 0,4 µl
Gibson 4: 1,4 µl, 1,4 µl, 0,1 µl
1h in 1 aliquot of Gibson mix (according to protocol)
→ pIG7002a, heat shock transfection in E. coli (according to protocol)
→ pIG7003a, heat shock transfection in E. coli (according to protocol)
→ pIG7004a, heat shock transfection in E. coli (according to protocol)

31.05.13

Digest of pIG7001a

used: colonie 1 (pIG7001a), NcoI, PstI HF, XbaI

µl type
5 DNA
1 NEB-Buffer 4
0,5 XbaI
0,1 BSA
Add to 10µl H2O
µl type
5 DNA
1 NEB-Buffer 4
0,5 NcoI HF
Add to 10µl H2O
µl type
5 DNA
1 NEB-Buffer 4
0,5 PstI HF
Add to 10µl H2O
  • Temp.: 37°C
  • Incubation time: 1h
expected bands
pIG7001a digest
left to right:
1: ladder (1kb)
2: XbaI
3: NcoI
4: PstI
5: negative control

XbaI did only cut once instead of twice, NcoI and PstI cut as expected

Sequencing of pIG7001a(1)

GATC-Watch-box: 706894
→ Sequence of pIG7001a ok

June

August

12.08.13

Digest of reporter plasmids

µl type
3 µg DNA (pKM602, pKM608, pKM611)
5 NEB-Buffer 4
1 Nhe I HF
1 Ssp I HF
0,5 BSA
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: 2h
µl type
3 µg DNA (pIG7001b, pIG7002b, pIG7004b)
5 Promega MC Buffer
1 Promega Nhe I
2 Promega Nru I
0.5 BSA
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: o/n

13.08.13

Gel run

A) Digest of pIG700..

From left to right: Marker (1 kb Roth); pIG7001; pIG7002; pIG7004 (50 & 4 µl); pIG7004 undigested (4 µl)
From left to right: Marker (1 kb Roth); pIG7001; pIG7002; pIG7004 (50 & 4 µl); pIG7004 undigested (4 µl)

B) Digest of pKM60..

From left to right: Marker (2 log NEB); pKM602; pKM608; pKM611 (50 & 4 µl)
From left to right: Marker (2 log NEB); pKM602; pKM608; pKM611 (50 & 4 µl)

All bands are at the expected sizes.

Gel extraction

DNA were purified using High Pure Plasmid Isolation Kit of Roche.

Changes to the protocol:

  • incubation at 56 °C for 10 min for gel dissolving
  • elution with dH2O (incubation at 50 °C for 4 min before centrifugation)

Yield: 10 ng/µl (pIG700..); 2 ng/µl (pKM60..)

Recombination of cutted parts

ingredient amount
pIG7001/2 2.5 µl
pKM602/11 2 µl
T4-Ligase 1 µl
T4-Ligase buffer 2 µl
dH2O up to 20 µl
Incubation at 22 °C for 1 h.
ingredient amount
pIG7004 4.3 µl
pKM608 1.2 µl
T4-Ligase 1 µl
T4-Ligase buffer 2 µl
dH2O up to 20 µl
Incubation at 22 °C for 1 h.

Trafo

  1. 4 µl of plasmid were added to 25 µl of chemically competent E. coli cells
  2. incubation for 10 min on ice
  3. heatshock (42 °C for 45 s)
  4. incubation for 2 min on ice
  5. addition of 300 µl LB medium
  6. incubation for 1 h at 37 °C (shaking)
  7. distribution of 300 µl on LB plates with kanamycin
  8. incubation over night at 37 °C

14.08.13

Picking of clones

From each Ligation 7 clones were picked and spread on 1/4 plates.

15.08.13

Miniprep

DNA was purified using High Pure Plasmid Isolation Kit of Roche.

Yield: ~ 220 ng/µl

Test digest

ingredient volume
plasmids (220 ng/µl) 1.2 µl
EcoRV-HF 0.5 µl
NEB buffer 4 1 µl
dH2O up to 10 µl
Incabation at 37 °C for 2 h.

Gel run

From left to right: Marker (1 kb Roth); pIG7005 (3x); pIG7006 (3x); pIG7007 (3x)

pIG7005_2, all clones of pIG7006 and pIG7007_1 & 3 showed the expected bands. pIG7005_2, pIG7006_1 and pIG7007_3 were send in for sequencing.

16.08.13

Midiprep

Though the sequencing did not have a result, plasmids were amplified and midiprepped by Pure Yield Plasmid Midiprep System from Promega because of the results of the test digest.

Seeding of cells for microscopy

A 24 well plate with cover slips was filled with 50,000 cells per well.

17.08.13

Transfection

Protocol:
  1. 40 µl Opti-MEM + 1.5 µl PEI-solution were mixed in a 1.5 ml Eppi.
  2. 0.5 µg of the DNA of interest were prepaired in another Eppi .
  3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
  4. Solution was spread drop-wise to the cells in the dish
Transfection scheme:

18.08.13

Fixation

Cells were washed with 500 µl PBS per well and fixated with 200 µl PFA per well (incubation on ice for 45 min).

Cover slips were washed in dH2O and mounted on a drop of Mowiol with DABCO on a object slide.

19.08.13

Flourescence microscopy

GFP and mCherry were expressed in all cells, only in the cells with TetR-VP16 there was a stronger signal. BFP was not detectable because of the microscope.

21.08.13

Seeding of cells for microscopy

A 24 well plate with cover slips was filled with 50,000 CHO cells per well.

22.08.13

Transfection

Protocol:
  1. 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
  2. 0.75 µg of the DNA of interest were prepaired in another Eppi .
  3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
  4. Solution was spread drop-wise to the cells in the dish

Medium change

Medium was changed after 3 h.

23.08.13

Fixation

Cells were washed with 500 µl PBS per well and fixated with 200 µl PFA per well (incubation on ice for 45 min).

Cover slips were washed in dH2O and mounted on a drop of Mowiol with DABCO on a object slide.

25.08.13

Flourescence microscopy

GFP and mCherry were detectable, but no differences in fluorescence intensity.

31.08.13

Seeding of cells for flow cytometry

A 24 well plate was filled with 50,000 HEK cells per well.

September

01.09.13

Transfection

Protocol:
  1. 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
  2. 0.75 µg of the DNA of interest were prepaired in another Eppi .
  3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
  4. Solution was spread drop-wise to the cells in the dish

DNA amount of effectors was 6 times higher than DNA amount of fluorescence proteins.

03.09.13

Preparation for flow cytometry

  • Removal of medium.
  • Washing with PBS.
  • Detaching of the cells with 25 µl trypsin per well.
  • Addition of 250 µl FACS buffer (PBS with 1 % FCS).
  • Samples were filled in little FACS tubes.

Flow cytometry

Intensities of all flourescent protein were measured.

Retrieved from "http://2013.igem.org/Team:Freiburg/Notebook/lab_multiple_targeting"