Team:Freiburg/Notebook/lab light

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Revision as of 23:26, 14 September 2013

Induction - Light

29.07.2013

Plasmids that we need as templates were transformed.

30.07.2013

Primers

Primers arrived today. Primers were diluted 1:10.

Minipreps

Transformed bacteria from yesterday with our template plasmids were minipreped.

pKM018: 83 ng/µl
pKM220: 216 ng/µl
pKM115: 177 ng/µl
pKM006: 118 ng/µl
pSAM200: 213 ng/µl
pSW42: 134 ng/µl
pSW45: 243 ng/µl
pIG2004: 212 ng/µl
T405: 213 ng/µl

Oligo Annealing of tetO crRNA

Oligos tetO13 1. CC and tetO13 2. CC were annealed
Therefore 20 µM diluted Primes were heated on 98° C for 4 minutes and then cooled down.

PCR list, program and composition

aIG4102: CMV Promotor from template T405 - Primers oIG4100F+R - 589 bp
aIG4103: Cas9 from template pIG2004 - Primers oIG4101F+R - 4170 bp
aIG4104: PIF and backbone from template pKM022- Primers oIG4102F+R - 3287 bp
aIG4105: KRAB from template pIG2004 - Primers oIG4103F+R - 390 bp
aIG4106: PhyB and backbone from template pKM018 - Primers oIG4104F+R - 4922 bp
aIG4107: UVR8 and backbone from template pKM168 - Primers oIG4105F+R - 3921 bp
aIG4108: COP1 and backbone frome template pKM115 - Primers oIG4106F+R - 3972 bp
aIG4109: CRY2 from template pSW42 - Primers oIG4107F+R - 1591 bp
aIG4110: Backbone from template pKM018 - Primers oIG4108F+R - 2924 bp
aIG4111: Backbone with VP16 from template pKM018 - Primers oIG4109F+R - 3282 bp
aIG4112: CIB from template pSW45 - Primers oIG4110F+R - 593 bp
aIG4113: Backbone from template pKM018 - Primers oIG4111F+R - 2881 bp
aIG4114: CIB from template pSW45 - Primers !! oIG4110F+oIG4112R - 638 bp

µl	Substance
10	Q5-HF Reaction Buffer
200 ng	Template
1	Primer forward
1	Primer reverse
4	dNTPs
1	DMSO
0,5	Q5-HF Polymerase
Add to 50	H2O

Temperature [° C]	Time
98	5 Min
98	30s
60 (or optimized)	30
72	30-40/kb
72	10 Min
4	hold

18 cycles

PCR results

aIG4102: 60° C - WORKED - GelExtraction: 87,9ng/ul
aIG4103: 60° C - 1 of 4 WORKED, Annealing Temperature lower? - GelExtraction: 50,5ng/ul Try 56°!
aIG4104: 60° C - DIDN'T WORK!!! Try with 58° - Was done, has to be tested on gel.
aIG4105: 60° C - WORKED - GelExtraction: 70,1ng/ul I and 57,7ng/ul II
aIG4106: 60° C - Was done, has to be tested on gel.
aIG4107: 60° C - ONLY SLIGHT BAND. TRY AGAIN!
aIG4108: 60° C - WRONG AMPLIFICATED BAND. Try again with different temperature!
aIG4109: 60° C - WORKED! - GelExtraction: 176,4ng/ul
aIG4110: 60° C - Was done, has to be tested on gel.
aIG4111: 60° C - WORKED! - GelExtraction: 84,4ng/ul
aIG4112: 60° C - Wasn't done yet.
aIG4113: 60° C - Was done, has to be tested on gel.
aIG4114: 60° C - Wan't done yet.

2-log Marker

aIG4102 (first 4) worked, 589 bp

aIG4105 (rest) worked, 390 bp

Roth Marker

aIG4103 only one PCR worked, 4170 bp

Roth Marker

aIG4104 didn't work

aIG4111 worked, 3282 bp

2-log Marker

aIG4109 (first two bands) worked, 1591 bp

aIG4107 (next two bands) didn't work, should have been 3921 bp

aIG4108 (one band) didnt work. Wrong band was amplified, should have been 3972 bp - another band of aIG4108 can be tested on a gel, didn't have space on this one.

31.07.13

PCR results

aIG4103: 54°-64° C - Everything WORKED!
aIG4104: 58° C - Gave three bands, upper band is supposed to be right. Was confirmed on a second gel with charging the gel only with a small amount of DNA.
aIG4106: 60° C - Didn't work. Try temperature gradient.
aIG4107: 54°-64° C - Everything Worked! But not very strong bands.
aIG4108: 54°-64° C - Didn't Work. Try seperate PCRs.
aIG4110: 60° C - Didn't work. Tried temperature gradient 54°-59° C - Worked with 55° and better with every additional ° C.
aIG4112: 60° C - Worked!
aIG4113: 60° C - Worked!
aIG4114: 60° C - Worked!

2-log marker

UPPER SIDE

aIG4103 (2 bands) - worked!

aIG4107 (2 bands) - worked!

aIG4106 (4 bands) - didn't work with 60° C, bands are too low!

LOWER SIDE

aIG4108 temperature gradient 54°-64° C - didn't work!

2-log marker

aIG4103 temperature gradient 54°-64° C - everything worked!

2-log marker

aIG4105 (2bands) - worked!

aIG4104 (2 bands) - upper band is considered to be the right band! - Worked!

aIG4110 - didn't work with 60° C.

2-log marker

aIG4107 temperature gradient 54°-64° C - worked, but only slight bands!

2-log marker

aIG4108 (2bands) - didn't work with 60° C!

aIG4113 (2 bands) - Worked!

aIG4110 - didn't work with 60° C.

2-log marker

aIG4110 (1 band) last column of temperature gradient (59° C) - worked!

aIG4112 (2 bands) - Worked!

aIG4114 - worked!.

2-log marker

aIG4110 temperature gradient (54°-59° C) - worked from 55° C upwards!

01.08.13

PCR results

aIG4104: Was gelexed and size was confirmed on a gel with a smaller amount of DNA. - Worked!
aIG4106: Temperature gradient 54° - 59° C and excess of reverse primer oIG4104R - Worked!
aIG4108: Seperate PCRs were tried out. Therefore the PCR was performed with template and at a time only one primer. After these PCRs (1x with forward Primer (58° C) and 1x with reverse Primer (62° C)) both columns were mixed and another PCR was performed. - Worked, but two bands appeared, upper band is considered to be the right fragment.

2-log marker

aIG4106 temperature gradient (54°-59° C) - worked!

2-log marker

aIG4108 seperate PCRs - worked, but other amplified products can be seen, too!

Gibsons

Fragments are mixed together to a total volume of 5µl and added to 15µl Gibson Master- Mix.

pIG4101: aIG4104- 0,83µl; aIG4103- 3,96µl; H2O- 0,21µl

pIG4103: aiG4107- 2,12µl; aiG4103- 2,88µl

pIG4105: aIG4110- 1,11µl; aIG4103- 3,51µl

pIG4106: aIG4111- 0,5µl; aIG4112- 0,3µl, H2O- 4,2ul

pIG4107: aIG4113- 0,4µl; aIG4105- 0,35µl; aIG4114- 0,35µl; H2O- 3,9µl

Gibson Mixes were transformed.

Ligation of pIG4100

Digest for pIG4100

µl	type
10	DNA (pKM006 or oIG4102) ~ 1000 ng
4	Cut Smart Buffer
1	EcoRI HF
1	Nhe1 HF
Add to 40µl	H₂O

Temp.: 37°C
Incubation time: 1,5h

2-log marker

Digested aIG4102 with 601 bp

Digested pKM006 with 6393 bp and 126 bp.

Both were gelexed: aIG4102 19 ng/µl and pKM006 40 ng/µl

Ligation and Transformation of approach pIG4100

µl	type
1	T4 Ligase
2	T4 Ligase buffer
3 fold molar amount of insert
2	Backbone pKM006
1,3	Insert aIG4102
13,7	H₂O

Temp.: RT
Incubation time: 15 minutes

After the ligation, 2,5 µl if this approach were transformed into E.coli and scratched out.

02.08.13

Gibson & Ligation results

Colonies could be seen on every plate except for one of the two ligation plates. Clones were picked and scratched out -> 51 Minis

Gibson

pIG4102: aIG4105- 0,4µl; aIG4106- 2,00µl; H2O- 2,6 µl

pIG4104: aiG4108- 3,2µl; aiG4105- 0,5µl; H2O- 1,3µl

Gibson Mixes were transformed.

03.08.13

Gibson results

Colonies could be seen on one of two plates, each. Colonies were scratched out for mini preps. Therefore we had clones for every construct.

Minipreps

51 Minis - concentrations between 50 and 200 ng/µl.

04.08.13

Minipreps

15 Minis.

05.08.13

Test digest

Today we made a test digest session of our 66 minis with following enzymes.

Plasmid	Enzymes	Size
pIG4100	EcoRI-HF & EcoRV-HF	1571 bp (Insert CMV 600bp) & 5417 bp
pIG4101	EcoRV-HF & NotI-HF	1952 bp (half the Cas9) & 5456 bp
pIG4102	EcoRI-HF & NotI-HF	1966 bp (with PhyB) & 3274 bp
pIG4103	BamHI-HF & XhoI	5337 bp (Cas9, UVR8) & 2682 bp
pIG4104	NotI-HF & BamHI-HF	1451 bp (with COP1) & 2839 bp
pIG4105	EcoRI-HF & EcoRV-HF	3040 bp (Half the Cas9 and half CRY2) & 5529 bp
pIG4106	NotI-HF & XbaI	970 bp (CIB, VP16) & 2833 bp
pIG4107	PstI-HF & NotI-HF	976 bp (CIB, KRAB) & 2821 bp

µl	type
~ 100-200 ng	DNA
1	Cut Smart buffer
0,5	Enzyme 1
0,5	Enzyme 2
Add to 10µl	H₂O

Temp.: 37°C
Incubation time: 2h

2-log marker

Upper bands: pIG4100, expected sizes: Plasmid 3 and 5 were send to GATC

Bottom bands: pIG4101, expected sizes: Plasmid 4 and 7 were send to GATC

2-log marker

pIG4102, expected sizes, Plasmid 1 and 9 were send to GATC.

2-log marker

Upper bands left side: pIG4104, expected sizes: Plasmid 1 and 4 were send to GATC

Upper bands right side: pIG4105, expected sizes: Plasmid 1 and 6 were send to GATC

2-log marker

Upper bands: pIG4106,
not expected sizes: Plasmid 3 was send to GATC

Bottom bands: pIG4107, expected sizes: Plasmid 1 and 4 were send to GATC

For the sequencing we used GATC Primers: CMV-F for pIG4100 and EBV-RP (binds in pSV40) for all other plasmids.

Insertion of crRNA into RNA Plasmid

First RNA Plasmid backbone was digested with BbsI for 3 hours. Enzyme was taken out of - 80° C.
Then it was put on a gel and gelexed. Concentration: 50,5ng/ug. Backbone from Hormone- Group: 8,7ng/ul
Ligation: Digested backbone from Hormone- Induction and self- digested backbone was used. Molar ratio of backbone : insert - 1 : 3.
Mix: Backbone; Insert, 1ul T4- ligase, 2ul T4-ligase-buffer, H2O up to a total volume of 20ul
Incubated for 15min at RT
Transformed and scratched out.

06.08

Sequencing results

Sequencing results were
positive for pIG4101, pIG4102, pIG4103, pIG4105, pIG4106 and pIG4107
(sequenced with EBV-RP -> SV40 Terminator). pIG4100 didn't have the CMV promotor as an insert (-> new ligation) and pIG4104 didn't contain KRAB, although KRAB worked with pIG4102 (new Primers?)

Re-Trafos and inoculation for midi-preps

The positive Plasmids and already existing plasmids pKM018 and pKM115 were retransformed and put into 150 ml LB with 150 µl Ampicillin and incubated at 37° C over night.

New Primers for pIG4104

New Primers were designed for pIG4104.

Ligation of pIG4100

Ligation of pIG4100 was done again like on 31.07.13, only the ratio of backbone and insert was changed (3 µl backbone and 1,5 µl Insert).

2-log marker

Left band: Backbone, Right band: Inser

Result of Ligation of RNA Plasmid

There were clones on every plate. They were scratched out for minipreps.

New sequencing

Plasmids were send to GATC with different primers. Cas9 was sequenced completely in pIG4101.7. Other pIG4104 were screened for insertion of KRAB.

7.8.2013

Midipreps of pKM017; pIG4101.7 (?), pIG4102.1, pIG4103, pKM115, pIG4105.5, pIG4106.3, pIG4107.4

All midipreps were successfull

Concentrations:

pKM018: 474 ng/ul

pIG4101: 485 ng/ul

pIG4102: 528 ng /ul

pIG4103: 521 ng/ul

pKM115: 517 ng/ul

pIG4105.1: 492 ng /ul

pIG4106: 567 ng/ul

pIG4107: 476 ng /ul

Minipreps of RNA Plasmid pIG4201 and pIG4202

Minis were send to GATC. 3 of ligation with digested Backbone from Michi and tetO1 and 3 with tetO2. And 3 of ligation with newly digested Backbone from Gabi and tetO1 and 3 with tetO2.

Result of Ligation of pIG4100

There were clones on every plate. They were scratched out for minis.

8.8.2013

Sequencing of RNA Plasmid

1 of the 6 pIG4201 and 4 of the 6 pIG4202 were positiv with inserted crRNA.

Sequencing Order

Sequencing of pIG4107_2 and pIG4107_3 with Primer EBV-RP

Minis of pIG4100

12 Minis were prepped.

Re-Trafo for inoculation for Midi Preps

The following plasmids were retransformed: pIG1000, pIG4201, pIG4202

9.8.2013

Midis of ...

Test digest of pIG4100

10.8.2013

Seeding of CHO cells

CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium

11.8.2013

Transfection of CHO cells

Protocol for 24- well plates:

1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi

2.Prepare in another Eppi 0.5 µg of the DNA of interest

3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT

4.Resuspend solution gently and spread them drop-wise to the cells in the dish

5.Swing the dish in axis-form

6.Change of media 5h post transfection

12.8.2013

PCB- treatment of CHO-cells and illumination

Cells are treated with HTS-medium containing 15uM of phycocyanobilin

illumination started at 16.15. with 20uE light- quantity

plate 1 with activating red light (660nm)

plate 2 with inctivating far-red light (740nm), control

construction of pIG4104

pKM115 was digested with BamHI and BssHII for 4h. KRAB was amplified from pIG2004 with oIG4103FII and oIG4103RII

Subsequently the fragments were assembled via Gibson-cloning and transformed into E.coli

12.8.2013

Gibson results

Cell-colonies are picked and plated out anew

A mistake in the cloning strategy was discovered, new primers are designed

13.8.2013

Gibson results

CHO- cells are illuminated

14.8.2013

Seeding of CHO-cells

CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium

SEAP-measurements of illuminated cells

The results are all negative except for the positive control (-> see transfection Nr.6 of 11.8.2013)

The interaction with the cri-RNA may be responsible. The experiment is repeated with new target- sites

15.8.2013

Transfection of CHO-cells for red light and UV tests

The wiki- transfection protocoll is used:

1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi

2.Prepare in another Eppi 0.5 µg of the DNA of interest

3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT

4.Resuspend solution gently and spread them drop-wise to the cells in the dish

5.Swing the dish in axis-form

6.Change of media 5h post transfection

The following pattern is transfected in 24- well plates:

16.8.2013

UV-b and red-/ far red light illumination of transfected cells from 15.8.2013

Cells for red- light illumination are illuminated with 20uE; 660nm or 720nm for 24h. Illumination is started at 14.20

Cells for UV-b light illumination are illiminated with 5uE; 311nm for 24h

17.8.2013

SEAP-measurements taken from illuminated- and untreated cells.

the following protocoll was obeyed for the whole process

200 µl of cell supernatant were transfered form the 24-well plate into one 96-well plate (round bottom)

Incubation for 30 min at 65 °C.

Centrifugation for 1 min at 1250 g.

100 µl of SEAP buffer were filled in the wells of a 96 well plate (flat bottom).

Addition of 80 µl supernatant from the round bottom plate to each well with SEAP buffer.

Addition of 20 µl pNPP (substrate).

96 plate was immediately placed in the blade reader.

Spectroscopic measurement every minute for 120 times (2h); wavelenght 405 nm.

numbers 1-6 are first six rows of the UV-b light approach -> look at transfections from 15.8.2013. numbers 7-12 are rows 1-6 of the red-light transfections from 15.8.2013

the system has to be improved, the controls showed the desired effect, but very weak. In the following the expression of Cas9- constructs has to be checked and improved. The tetO cri-RNA showed an insufficient binding pattern. New target sites for the plasmid have to be construct

20.8.2013

Construction of new cri-RNAs

6 new cri-RNAs are designed and ordered at sigma-aldrich. One new cri-RNA is provided by Max Stamnitz. All new cri-RNAs target pKM006!

Cloning of pIG4100; pIG4104; pIG4106; pIG4107

Primers for new cloning- strategies have arrived.

pIG4100: pKM006 is digested and the CMV-promotor from pAcGFP1 is amplified via PCR.

pIG4104: pKM115 is digested with BamHI and BssHII. KRAB is amplified via PCR from pIG2001

pIG4106: pKM018 is digested with EcoRI and NotI. CIB is amplified from pSW45

pIG4107: pIG4102 is digested with EcoRI and NotI. CIB is amplified from pSW45

21.8.2013

Cloning of pIG4100; pIG4104; pIG4106; pIG4107

Products from digestions and PCRs are purified via gel run in 1% agarose and gelexed The digest of pKM115 for pIG4104 has not worked and is repeated executing the same protocol.

Parts for pIG4100, pIG4106, pIG4107 are Gibson cloned and subsequently transformated into competent "TOP10" E.coli- cells

22.8.2013

Cloning of pIG4100; pIG4104; pIG4106; pIG4107

Clones of Gibsons are picked and plated out. Later to be picked and test- digested. For each plasmid 2 samples were sent to GATC for sequencing.

Gibson- cloning of pIG4104 is repeated with new digest and PCR-fragments. The constructs are subsequently transformated into competent "TOP10" E.coli- cells

Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic?

HEK, HeLa and CHO- cells are seeded in 6-well plates

23.8.2013

Cloning of pIG4104

Colonies are picked and plated out for mini and test- digest on 24.- or 25.8.13

Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic?

different DNA- concentrations are used for transfection in different cell lines. pIG1000 containing Cas9-VP16 is used. As a already tested sample, pIG8004 containing Cas9-G9a is used as positive- control. The following DNA- concentrations are transfected: 2ug; 3ug; 4ug; 6ug. One sample is tested with and addition of 0,5% DMSO into the medium attempting to boost the expression

The following pattern is transfected

24.8.2013

Oligo annealing

Targets T2, T3, T4, T5, T6, T7 and Max's random target were annealed.

Ligation of new RNA plasmid

Digested pIG3010 (50,1 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert.

25.8.2013

Result of ligation

Colonies were picked for mini preps.

26.8.2013

Minipreps of targets

Minipreps were sent for sequencing

27.8.2013

Result of sequencing

T2, T5 and T6 were positive.

Seeding of cells for UV light activation experiment

29.8.2013

Transfection of CHOs for UV light activation

30.8.2013

Light start

UV light was started at 15.30 o clock (two plexi glass panes)

31.8.2013

SEAP assay

SEAP assay of UV light activation was performed.

01.9.2013

02.9.2013

03.9.2013

Oligo annealing

5µl Oligo forward 100 µM to get 20 µM concentration
5 µl Oligo reverse 100 µM to get 20 µM concentration
10 µl NEB buffer 2
30 µl ddH2O

Mix was heated to 98° C, Heatblock was turned off and Oligos were cooled down

Digest of pIG3010

10 µl pIG3010
1 µl BbsI from -80° C
5 µl NEB buffer 2.1
up to 50 µl ddH2O

Digestion was put on a gel and purified (10 ng/µl).

Ligation of Target 3,4 and VEGF and transformation

Digested pIG3010 (10 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert.

04.9.2013

Result of Ligation

Colonies were picked

Gibson of pIG4104

Wrong primers were used for the last pIG4104 plasmid (was seen with the last sequencing). Therefore we made a new Gibson approach.

05.9.2013

Minipreps of T3, T4 and VEGF (plates) and pIG4104 (fluid culture)

Minipreps were send for sequencing (Primers for RNA Plasmid (in pIG3010) BGH-reverse and EBV-RP for pIG4104.)

06.9.2013

Sequencing results

pIG4104-3 was positive, T3, T4 and VEGF RNA plasmids were negative.

New Ligation of T4 target into standardized RNA plasmid

Procedure was done by standard protocol.

Seeding of CHO cells

4 24-well plates were ceeded with 65000 CHO cells per well.

07.9.2013

Status of CHO cells

CHO cells were all contaminated with bacteria again. Media was plated in a 10 cm dish. Result: No contamination here.

seeding of HEK cells

HEK cells were seeded for UV & BL activation and repression experiments at around 19/19.30. 4 24-well plates and 65000 cells per well.

08.09.2013

Transfection of HEKs for UV & BL activation and repression

HEK cells were transfected (standard protocol) at 19.30. Media was changed after 5 hours.

T4 RNA plasmid

6 colonies were scratched out.

09.09.2013

Minis of T4

3 of 6 plasmids were sent for sequencing (1-3).

Light start

Blue light was started at 21.30 with 450 nm and unsure intesity (4 colour box, 655 intensity number). Dark control is in a light box without light on.

UV light was started at 20.30 Dark control is in the incubator (no UV light in the lab).

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-						<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375395279&w=500&h=376&media=wiki:laborbuecher:07-31_aig4107_temperaturgradient.jpg"> </td>
+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/d/d9/8light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/8/8b/9light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/2/22/10light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/b/b9/11light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/b/b5/12light2_0_freiburg_13.jpg"> </td>
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-						<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375396754&w=500&h=376&media=wiki:laborbuecher:08-01_aig4108.jpg"> </td>
+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/3/39/13light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/7/75/15light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/7/70/16light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/f/f8/17light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/d/dc/18light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/c/cb/19light2_0_freiburg_13.jpg"> </td>
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+						<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/8/89/20light2_0_freiburg_13.jpg"> </td>
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         <h2> 11.8.2013 </h2>
         <h3> Transfection of CHO cells </h3>
-            <td>  <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=1.jpg> </td>
+            <td>  <img class="gelpic" src="https://static.igem.org/mediawiki/2013/2/22/21light2_0_freiburg_13.jpg"> </td>
-            <td>  <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=2.jpg> </td>
+            <td>  <img class="gelpic" src="https://static.igem.org/mediawiki/2013/d/d1/22light2_0_freiburg_13.jpg" </td>
-            <td>  <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376472011&w=500&h=404&media=wiki:laborbuecher:licht20:11-08_1_.jpg </td>
+            <td>  <img class="gelpic" src="https://static.igem.org/mediawiki/2013/a/a0/23light2_0_freiburg_13.jpg" </td>
-           <td>  <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376472011&w=500&h=404&media=wiki:laborbuecher:licht20:11-08_2_.jpg </td>
+<td>  <img class="gelpic" src="https://static.igem.org/mediawiki/2013/d/d0/24light2_0_freiburg_13.jpg" </td>
         <h4> Protocol for 24- well plates: </h4>
             <p> 1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi </p>
@@ Line 723: / Line 723: @@
                    <p> The following pattern is transfected in 24- well plates: </p>
-                      <td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=wiki:laborbuecher:licht20:15-08_uv.jpg> </td>
+                      <td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/8/8d/25light2_0_freiburg_13.jpg"> </td>
-                      <td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=wiki:laborbuecher:licht20:15-08_rot_660nm.jpg> </td>
+                      <td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/5/51/26light2_0_freiburg_13.jpg"> </td>
-                      <td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=wiki:laborbuecher:licht20:15-08_rot_740nm.jpg> </td>
+                      <td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/3/3b/27light2_0_freiburg_13.jpg"> </td>
              <h2> 16.8.2013 </h2>
@@ Line 745: / Line 745: @@
                             <p>Spectroscopic measurement every minute for 120 times (2h); wavelenght 405 nm.</p>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378423063&w=500&h=325&media=wiki:laborbuecher:licht20:rl_17-08.png> </td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/0/02/28light2_0_freiburg_13.png"> </td>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378423064&w=500&h=340&media=wiki:laborbuecher:licht20:uv_17-08.png> </td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/e/e6/29light2_0_freiburg_13.png"> </td>
                      <p> numbers 1-6 are first six rows of the UV-b light approach -> look at transfections from 15.8.2013. numbers 7-12 are rows 1-6 of the red-light transfections from 15.8.2013 </p>
@@ Line 784: / Line 784: @@
                         <p> different DNA- concentrations are used for transfection in different cell lines. pIG1000 containing Cas9-VP16 is used. As a already tested sample, pIG8004 containing Cas9-G9a is used as positive- control. The following DNA- concentrations are transfected: 2ug; 3ug; 4ug; 6ug. One sample is tested with and addition of 0,5% DMSO into the medium attempting to boost the expression </p>
                         <p> The following pattern is transfected </p>
-                        <td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=wiki:laborbuecher:licht20:123.png> </td>
+                        <td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/b/bf/30light2_0_freiburg_13.png"> </td>
 		<h2> 24.8.2013 </h2>
 			<h3> Oligo annealing </h3>
 			<p> Targets T2, T3, T4, T5, T6, T7 and Max's random target were annealed.</p>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378423065&w=500&h=264&media=wiki:laborbuecher:licht20:light_targets.png> </td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/c/c7/31light2_0_freiburg_13.png"> </td>
 			<h3> Ligation of new RNA plasmid </h3>
 			<p> Digested pIG3010 (50,1 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert. </p>
@@ Line 805: / Line 805: @@
 			<h3> Transfection of CHOs for UV light activation</h3>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378422527&w=436&h=468&media=wiki:laborbuecher:licht20:uv_light_transfection_27-08.png> </td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/f/f8/32light2_0_freiburg_13.png"> </td>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378422528&w=500&h=393&media=wiki:laborbuecher:licht20:uv_transfection_calc1_27-08.png> </td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/f/f4/33light2_0_freiburg_13.png"> </td>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378422528&w=500&h=355&media=wiki:laborbuecher:licht20:uv_transfection_calc2_27-08.png> </td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/c/c6/34light2_0_freiburg_13.png"> </td>
 		<h2> 30.8.2013 </h2>
@@ Line 817: / Line 817: @@
 			<h3> SEAP assay </h3>
 			<p> SEAP assay of UV light activation was performed. </p>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378423064&w=499&h=255&media=wiki:laborbuecher:licht20:uv_31-08.png> </td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/b/b4/35light2_0_freiburg_13.png"> </td>
 		<h2> 01.9.2013 </h2>
 			<h3> </h3>
@@ Line 864: / Line 864: @@
 			<h3> Transfection of HEKs for UV & BL activation and repression</h3>
 			<p> HEK cells were transfected (standard protocol) at 19.30. Media was changed after 5 hours.</p>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=499&h=366&media=wiki:laborbuecher:licht20:bl_transfection_plan_08-09.jpg></td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/2/23/36light2_0_freiburg_13.jpg"></td>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=500&h=371&media=wiki:laborbuecher:licht20:uv_transfection_plan_08-09.jpg></td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/f/ff/37light2_0_freiburg_13.jpg"></td>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=499&h=392&media=wiki:laborbuecher:licht20:bl_transfection_calc_08-09_1.jpg></td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/5/55/38light2_0_freiburg_13.jpg"></td>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=499&h=356&media=wiki:laborbuecher:licht20:bl_transfection_calc_08-09_2.jpg></td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/3/3e/39light2_0_freiburg_13.jpg"></td>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=500&h=388&media=wiki:laborbuecher:licht20:uv_transfection_calc_08-09_1.jpg></td>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/8/86/40light2_0_freiburg_13.jpg"></td>
-<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=499&h=354&media=wiki:laborbuecher:licht20:uv_transfection_calc_08-09_2.jpg></td>			<h3> T4 RNA plasmid</h3>
+<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/4/4d/41light2_0_freiburg_13.jpg"></td>			<h3> T4 RNA plasmid</h3>
 			<p> 6 colonies were scratched out. </p>
 		<h2> 09.09.2013 </h2>