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Team:Freiburg/Notebook/lab multiple targeting
From 2013.igem.org
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
| 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) |
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 |
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 |
| ingredient | amount |
|---|---|
| pIG7004 | 4.3 µl |
| pKM608 | 1.2 µl |
| T4-Ligase | 1 µl |
| T4-Ligase buffer | 2 µl |
| dH2O | up to 20 µl |
Trafo
- 4 µl of plasmid were added to 25 µl of chemically competent E. coli cells
- incubation for 10 min on ice
- heatshock (42 °C for 45 s)
- incubation for 2 min on ice
- addition of 300 µl LB medium
- incubation for 1 h at 37 °C (shaking)
- distribution of 300 µl on LB plates with kanamycin
- 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 |
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
- 40 µl Opti-MEM + 1.5 µl PEI-solution were mixed in a 1.5 ml Eppi.
- 0.5 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
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
- 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 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
- 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
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.
