Team:Potsdam/notebook/LLPS

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Lab book

We separated our lab book into the following parts:

Monday, 08/21/17

- prepared 500 ml of synthetic dropout (SD) medium:

- 3,35 g nitrogen base + 0,39 g dropout mix diluted in 450 ml water

- adjusted pH to 5,75 with 1 M NaOH

- adjusted volume to 475 ml with water

- filled in a 1l-bottle with 10 g agar

- autoclave

- prepared a 40% glucose solution (500 ml)

-> autoclave separately and add 25 ml to SD medium before use!

- streaked yeast strain YPH500 on YPDA plates (on clean bench)

incubation for 2,5 days at 30°C

Tuesday, 08/22/17

- transformation of 1 ng pYES-CT2 into competent E. coli cells

plated 200 µl of the transformation on amp plate
incubation over night at 37°C

Wednesday, 08/23/17

- transformation hasn't worked

trafo was done again: new aliquot of pYES2/CT (128 ng/ul)

- two transformations into competent JM109: 1 ul of aliquot, 1 ul of 1:10 dilution
- plated 50 µl of each transformation mix on amp plates, centrifuged the remaining transformation mix, resuspended in residual medium and plated on additional amp plates

- Amplification of IDT- fragments (see low-copy lab book)

- Eppi: "D", 65°C annealling Temp.

- Primer: Q35/36

- tomorrow test gel run

- inoculation of YPH500 (one clone from the plate from monday (21.08.17)) in 10 ml YPD-Plus-Medium (from zymo research) for production of competent cells

- incubation over night at 30°C and shaking at 200 rpm

Thursday, 08/24/17

- culture did not grow (not enough cells, use inoculation needle!)
- new culture made for competent cells
- culture for miniprep of pYES vector for tomorrow (no colonies on yesterday's plate)
- results and proceedings of test gel for Ddx4-YFP under low-copy lab book

Friday, 08/25/17

- miniprep (pYES Vector)
- Glycerolstock:

80% Glycerol	0,3 mL
Miniprep	0,5 mL

Test digestion:

total volume	10 µL
Miniprep (pYES)	2 µL (=324,8 ng)
PST1	0,2 µL
Buffer 3.1	1 µL
ddH<sub>2</sub>O	6,8 µL

- 2h at 37°C
- run gel for 30 min and 100V
- Result: 3 bands

- 400-500 (actually: 488bp)

- 2000-3000 (actually: 2291bp)

- 3000-4000 (actually: 3184bp)

- Dagmar added EtBr to 1% Agarose

Monday, 08/28/17

- preparative digest of pYES_2CT

total volume	30 µL
EcoRI-HF	1 µL
XbaI	1 µL
Cutsmart	3 µL
ddH<sub>2</sub>O	10 µL
Miniprep (pYES2_CT)	15 µL

- incubate for 2h at 37° shaking
- heat inactivation at 80° for 20 min (no shaking)
- PCR clean up according to Promega kit (stored in our -20° freezer, concentration 51,8 ng/µl)
- inoculated preculture for competent cells (yeast, in right 30° shaker)

Tuesday, 08/29/17

-OD of yeast culture from yesterday: 1,15

-> dilution: 2 ml of culture and 8 ml YPDA medium

-culture stopped growing (probably because of change of medium, always use YPDA medium for cultures. Our medium is only for transformations -> expensive)
-inoculated new culture (15 ml YPDA medium) -> in 30° shaker

Wednesday, 08/30/17

-still working on competent cells: OD600 this morning not measurable
-diluted twice: first 2 ml yeast-culture + 8 ml medium -> OD still too high; again diluted 1/5 (2 ml yeast-culture + 8 ml medium) -> final dilution factor 1/25
-harvested culture at OD=1,18
-because of OD measurements only 9 ml cultur left -> added 1 ml medium (so we could use the 10 ml tara)
-for procedure see protocol of ZymoResearch on folder
-We use the kit in the fridge.
-use a 10 ml Tara for centrifuging
-cells stored in -80° freezer in a box labelled with LLPS, tubes labelled with CY (Competent Yeast)

Thursday, 08/31/17

-produced plates (Dropout-Uracil, added 25 ml of 40% Glucose)
-transformation of pYES and Ddx4YFP in competent yeast cells:
-50 µl competent cells, 100 ng pYES (digested), 100 ng Ddx4YFP
-incubated 45 min at 30°C and 300 rpm
-plated 150 µl and 50 µl

Monday, 09/04/17

-did transformation of pYES and Ddx4-YFP again because last time not done under steril conditions (no yeast clones on plates, only contamination last time):

added 5,1 µl of Ddx4-YFP (tube "YFP 6", c = 19,6 ng/µl)

added 1,96 µl of digested pYES (c = 51 ng/µl)

added 500 µl EZ3 solution

to 50 µl competent yeast cells

-plated on yeast plates as 50 µ / 150 µl
-incubation 2-3 days at 30°C

Wednesday, 09/06/17

-plating out 8 clones from the Trafo plate (04.09.17; 150ul) onto a master plate: clean bench with inoculation loops

Friday, 09/08/17

-colony PCR for yeast with 8 clones from the master plate
-resuspend clones with 0,02 M NaOH (clean bench, black clones)
Mastermix:

10µl Primer 1 (IG_X_Q25)

10µl Primer 2 (IG_X_Q26)

80µl ddH2O

125µl ALLin™ Red Taq MasterMix, 2X

22,5µl MasterMix +2,5µl resuspended cells

5min incubation

PCR

->Annealing temperature: 61°C, elongation time: 28s
-Gel Electrophoresis: all clones negativ
-second colony PCR:
-resuspend clones with 0,02 M NaOH (clean bench, red clones)

22,5µl MasterMix +2,5µl resuspended cells

7min incubation

Monday, 09/11/17

-loaded colony PCR products from friday on a 1% agarose gel (100 V, 45 min)

clone nr. 1-8 (nr. 4 accidentally picked two times)

'-'= negative control (only water)

clone nr. 2, 3, 5, 7 and 8 showing correct band (~ 1900 bp)

-picked clone nr. 2, 3, 5, 7 and 8 again from master plate and plated on new SD-Ura plates + inoculation of the same clones in 3 ml SD-Ura liquid medium (incubation 30°C, + liquid culture shaking with 200 rpm)
-also picked a YPH500 clone (plate from 21.08.17) and plated on new SD-Ura plate -> will not grow! (should have used YPDA medium!) + inoculation of the clone in 3 ml SD-Ura liquid medium -> will also not grow

Tuesday, 09/12/17

-liquid culture from yesterday did not grow -> discarded
-same clones on the new SD-Ura plates also did not grow -> spreaded them a little more with the loop
-not enough growth til afternoon -> have to grow over night
-make new liquid culture tomorrow (better: 5 ml liquid cultere in 100 ml flask) for microscopy on thursday
-clone 8 didnt grow at all -> discarded
-made YPDA medium (1x500 ml for plates (with agar) and 1x500 ml for liquid culture (without agar; in cold room):

10 g Difco peptone

5 g yeast extract

(10 g agar) -> only for plates

15 mg adenine hydrochloride

(we didnt adjust the pH -> Fabian said we dont have to)

-> add 25 ml 40% glucose before use!

-made YPDA plates -> cold room
-plated YPH500 from old plate (21.08.17) on new YPDA plate -> incubation at 30°C
-made SD-Ura medium (liquid) -> cold room

Wednesday, 09/13/17

-YPH500-Ddx4-YFP clones (2, 3, 5 and 7) on SD-Ura plates -> sufficiently grown
-made 6 ml liquid culture (SD-Ura medium + 25 ml 40% glucose) for tomorrow
-transferred quite a lot of cell material from plate to liquid medium (with the loop) -> significant clouding of liquid
-used baffled flasks for better growth
-additionall inoculation of YPH500 (wild type) as control for microscopy
-made 6 ml liquid culture (YPDA medium + 25 ml 40% glucose)
-transferred quite a lot cell material from plate (21.08.17) to liquid medium (with the loop) -> significant clouding of liquid
-used baffled flasks for better growth
-For tomorrow: please take the new plate of YPH500 out of the incubator -> use parafilm to protect the plate from drying out

Thursday, 09/14/17

-WT from 13.9. totally overgrown -> next time more medium/less cell material
-Results of fluorescence microscopy:

fluorescence visible

"noisy" background fluorescence in WT yeast

Addition of NaCl intensified background fluorescence in WT (we have no explanation for that yet)

-->Neither change in temperature nor in osmolarity induced the formation of droplets
-> Not possible though in this experiment, because the plasmid contains a Galactose inducible promoter and no Galactose was added
On the contrary: The medium used contained Glucose, which represses the translation of the Ddx4-YFP construct
-Started preparing SD medium (800 ml H2O+ 6,7 g Yeast Nitrogen Bas + Aminoacid mix) -> sent for autoclaving

Wednesday, 09/20/17

-preparation of preculture for induction culture

wild type	clone 2	clone 3	clone 5	clone 7
15ml YPDA Media	15ml SD-Ura Media	15ml SD-Ura Media	15ml SD-Ura Media	15ml SD-Ura Media

-grow at 30°C over night (till 5PM 21.09.)

Thursday, 09/21/17

-6PM: prepared induction culture (for fluorescence microscopy tomorrow)
-determined the OD(600) of the preculture (20.09.)
-calculated amount of preculture necessary to obtain an OD(600) of 0,4 in 10ml of induction media:
-->(0,4 OD*ml-1*10ml)/measured OD*ml-1 (results in table 3)

Clone	Wild type	clone 2	clone 3	clone 5	clone 7
OD	9,0	5,39	5,29	5,18	5,66
Volume preculture	0,45ml	0,74ml	0,75ml	0,77ml	0,70ml

-removed the amount of preculture into an Eppi and centrifuged for 5min at 1500xg
-discarded supernatant
-resuspended the pellet with 10ml induction media (14.09.)
-incubated for 16h at 30°C with shaking

Friday, 09/22/17

-flourescence microscopy in house 20:
-only less fluorescence visible in all samples (wt, clones with Ddx4-YFP in galactose or glucose SD-Ura medium)
-it seemed like all clones didnt grow well in the medium with galactose -> less cells and most of them still in cell division phase

-fluorescence microscopy at the mpi: used leica confocal microscope (software for the pictures: LAS AF)

Induction with Galactose	clone	file (# picture)	Comment
+	7	3-11	positive! in average 1 droplet per cell, sometimes even more (2-3)
-	7	13-15	no fluorescence, only during cell division
+	5	19	no fluorescence, only during cell division
-	5	17
+	3	21	no fluorescence, only during cell division
-	3	23
+	2	rest	positive!
-	2	25	no fluorescence, only during cell division
-	wild type	34	no fluorescence, only during cell division

-> positive clones: 2 and 7
-droplets were visible without induction with cold shock or osmotic shock!
-->(because of high Ddx4-YFP concentrations in yeast)
-->incubation on ice (cold shock) for ~ 1 min -> had no effect on droplets
incubation on a ~ 37°C block (heat shock) for ~ 1 min:
-->droplets partially disappeared -> more smaller droplets, less intense when cooling down again
-->fluorescence spread over entire cell

Monday, 09/25/17

-YPH500-Ddx4-YFP clone 2 and 7 (plate of September 11th) newly streaked on SD-Ura plate (two on one plate)
-> for glycerol stocks - have to incubate for two days at 30°
-prepared SD-Ura medium (Raffinose+Galactose+Tryptohane)

ingredients	for plates (500 ml)	for liquid medium
yeast nitrogen base	3,35 g	6,7 g
dropout mix	0,39 g	0,77 g
Tryptophane	2 g	4 g
water	400 ml	800 ml

-adjusted pH to 5,6 (plates) and 5,7 (liquid)
-sent to autoclave
-added (sterile filtered!)

	for plates	for liquid medium
galactose (20%)	50 ml	100 ml
Raffinose (10%)	50 ml	100 ml

-Medium for plates has a really weird colour, will be done again tomorrow!

Tuesday, 09/26/17

-repetition SD-Ura + Trp media for plates (500ml), composition like yesterday, plates poured
-Digestion of pYES 12 CT with PmeI and HindIII-H7

->2µl plasmid (1 ng/µl) + 1µl HindIII-H7 + 1µl PmeI + 3µl 10x CutSmart + 23µl water

incubation at 37°C for 3h 45min

heat inactivation at 80°C for 20 min

-preparation gel: 0,7%

-30µl digest + 6µl LD

-> no bands visible

-Hypothesis: no DNA in pYES12CT 1ng/µl (to little DNA -> can not be detected in the gel)
-new digestion over night (same preparation as before, only difference: 2,5µl plasmid 1ng/µl; 22,5µl water)
-> to litte DNA, too see gel picture in low copy)

Wednesday, 09/27/17

-Digest pYES2-CT repeated
-pYES 25.08.17; 162,4ng/µl

12,3µl pYES2-CT (2ng) + 3µl Cutsmart + 12,7µl water + 1µl HindIII-HF + 1µl PmeI

Incubation: 1,5h at 37°C

digestion on 0,7% agarose gel

-> 50 min at 120V

-Expected bands: 5782bp (backbone purified from gel) + 181bp (not visible, possibly to small)
-Gel clean-up with Promega-Kit
-concentration: 28,2ng/µl
-Ddx4-IAAH and Ddx4-IAAM from IDT in 20µl TE-Buffer resuspended
-final concentration: 50ng/µl

Thursday, 09/28/17

-Glycerol stocks of YPH500-Ddx4-YFP clone 2 and 7
-resuspended cells in 355µl SD-Ura-Media (+Glucose)
-added 145µl of sterile glycerol (86%)
->stored at -80°C

Friday, 09/29/17

-Transformation

50µl competent yeast cells

100 ng (3,55µl) pYES (Hind3, Pmel1)

100 ng (2µl) dDx4-IAAH

100 ng (2µl) dDx4-IAAM

-PCR
1) Ddx-YFP

2,5µl IG_X_Q100 (10µM)

2,5µl IG_X_Q101 (10µM)

19µl ddH2O

25µl MM Q5

1µl Template

2) Ddx-IAAM

1,25µl IG_X_Q103 (10µM)

2,5µl IG_X_Q104 (10µM)

9,5µl ddH2O

12,5µl Master Mix Q5

1µl Template

3) Ddx-IAAH

1,25µl IG_X_Q105 (10µM)

1,25µl IG_X_Q106 (10µM)

9,5µl ddH2O

12,5µl Master Mix Q5

1µl Template

4) Ddx-IAAM

1,25µl IG_X_Q112 (10µM)

1,25µl IG_X_Q113 (10µM)

9,5µl ddH2O

12,5µl Master Mix Q5

1µl Template

5) Ddx-IAAH

1,25µl IG_X_Q114 (10µM)

1,25µl IG_X_Q115 (10µM)

9,5µl ddH2O

12,5µl Master Mix Q5

1µl Template

approach	initial denaturation	Denaturation	Annealing	Extension	Final Extension	Hold
1	98°C 30sec	98°C 10sec	57,9°C 15sec	72°C 15sec	72°C 5,6min	4°C
2	98°C 30sec	98°C 10sec	62,6°C 15sec	72°C 15sec	72°C 5,6min	4°C
3	98°C 30sec	98°C 10sec	68°C 15sec	72°C 15sec	72°C 5,6min	4°C
4	98°C 30sec	98°C 10sec	64,2°C 15sec	72°C 15sec	72°C 5,6min	4°C
5	98°C 30sec	98°C 10sec	57,9°C 15sec	72°C 15sec	72°C 5,6min	4°C

Monday, 10/02/17

-run a 1% agarose gel (40 min, 120 V) withe the rest of the PCR products from 29.09.17
-Gel clean-up:

PCR product	size (bp)	concentration (ng/µl)
Ddx4-YFP with BioBrick extensions	1765	44.0
Ddx4-IAAM (IDT)	2839	8.7
Ddx4-IAAH (IDT)	2871	9.7
CDS-IAAH	1410	52.5
CDS-IAAM	1704	59.8

-PCR to amplify the IDT fragments (# 2 and 3 in table8) needs to be optimized (gradient PCR + different dilutions)
-Digestion of Ddx4-YFP with BioBrick extensions with EcoRI-HF and PstI

component	volume (µl)
NEBuffer 2.1	1
EcoRI-HF	0.5
PstI	0.5
water	1.2
Ddx4-YFT with BB (44 ng/µl) -> used 300 ng	6.8

-->incubation: 1 h, 37°C
-->heat inactivation: 20 min, 80°C
-Ligation of digested Ddx4-YFP with BB with pSB1C3

component	volume (µl)
T4 DNA Ligase Buffer	2
T4 DNA Ligase	1
pSB1C3 (12.5 ng/µl)-> used 50 ng	4
water	8.6
Ddx4-YFT with BB_d E+P -> used 132.4 ng	4.4
Ddx4-YFT with BB (44 ng/µl) -> used 300 ng	6.8

-->incubation: over night, 16°C
-Gibson Assemblies for LLPS control: insert CDS of IAAM/H into pYES2_CT_d_PmeI+HindIII

component	volume (µl)	volume (µl)	volume (µl)	volume (µl)
Vector: pYES_d	1.8	5782	28.2	50
Insert: CDS-IAAM	0.5	1700	59.8	29.4
water	7.7	-	-	-
Master Mix	10	-	-	-

-->Incubation: 19 min, 50°C

component	volume (µl)	volume (µl)	volume (µl)	volume (µl)
Vector: pYES_d	1.8	5782	28.2	50
Insert: CDS-IAAH	2.2	1400	52.5	24.2
water	6	-	-	-
Master Mix	10	-	-	-

-->Incubation: 15 min, 50°C
-transformation of 5 µl Assembly product into DH5a
-plated on amp plates
-Gibson Assembly for LLPS main part (plan B if TAR did'nt work): Fusion of Ddx4-IAAM (IDT) + Ddx4-IAAH (IDT)

component	volume (µl)	volume (µl)	volume (µl)	volume (µl)
Ddx4-IAAM (IDT)	1	50	50	50
Ddx4-IAAH (IDT)	1	50	50	24.2
water	8	-	-	-
Master Mix	10	-	-	-

-->Incubation: 25 min, 50°C
-stored at -20°C
-made 2 master plates of yeast transformation (29.09.17): 16 clones
-->incubation 2-3 days at 30°C
-The CDS IAAM and CDS-IAAH are send for sequencing:

Tuesday, 10/03/17

-colony-PCR for

CDS-IAAM (primer: IG_X_Q25 and IG_X_Q116) in pYES and

CDS-IAAH (primer: IG_X_Q25 and IG_x_Q117) in pYes

from Gibson-Assembly 02.10.17 -run 10 µl PCR-reaction on 0,7 % agarose gel -PCR-program:

initial denaturation: 1 min 95 °C

denaturation: 15s 95 °C

annealing: 15 s 56 °C

extension: 17 s 72°C

final extension: 5 min 72 °C

PCR Ddx4-IAAM-Ddx4-IAAH (after Gibson-Assembly from 02.10.17):
-3 aliquotes (à 50 µl):

- undiluted Gibson-Assembly

- dilution 1:10 Gibson-Assembly

- dilution 1:100 Gibson-Assembly

Mastermix (3x);

component
Taq Mastermix (Q5 Mastermix should have been used)	75
IG_X_Q103	7.5
IG_X_Q106	7.5
ddH20	57

-each: 49 µl Mastermix + 1 µl template
PCR-program:

initial denaturation: 98 °C 30s

denaturation: 98 °C 10s

annealing: 63°C 30 s

extension: 72 °C 171 s

final extension: 72 °C 2 min

35 cycles

-each: 2 µl + 8 µl ddH2O
-1:10 and 1:100 -> 2 µl loading dye
-run on 0,7 % agarose gel
->result: no correct bands (5680 bp) -> gradient-PCR
-gradient-PCR:
-for undiluted Gibson-Assembly, dilution 1:10 and dilution 1:100 each 6 different annealing temperature:

58 °C

58,9 °C

61,1 °C

64,9 °C

67,1 °C

68 °C

each:

component
Q5 Mastermix	5
IG_X_Q103	0.5
IG_X_Q106	0.5
ddH20	3
template	1

-transformation: Ddx4-YFP in psB1C3 in E.coli cells -miniprep with the following clones: -->CDS-IAAH: 2,7,8,9,10
-->CDS-IAAM: 3,5,6,7,8
Wednesday, 10/04/17

-test gel of gradient PCR (Gibson of Ddx4-IAAM-Ddx4-IAAH) of October 3rd
-on gel: undiluted DNA (Ddx4-IAAM-Ddx4-IAAH), 1;10 diluted DNA, 1:100 diluted DNA with the annealing temperatures 58°, 59°, 61°, 65° and 68°
-> result: only the 1:10 diluted samples at 58°, 59° and 61° show correct bands (~5700 bp), but also other bands
-repeated PCR with 1:10 diluted template at the annnealing temperatures 58°, 59° and 61° in 50 µl batches
-for three reactions:

component	volume (µl)
Q5 Mastermix	75 µl
IG_X_Q123	7,5 µl
IG_X_Q106	7,5 µl
ddH20	57 µl
template (per reaction)	1 µl

-49 µl mix per tube + 1 µl template (1:10 Ddx4-IAAM-Ddx4-IAAH)
-PCR program see October 3rd, annealing temperatures now are 58°, 59° and 61°
--> actually wanted to get more of the corrrect DNA to extract it from a gel
-gel picture: negative! No corrrect bands! (No extraction done)
-assumption: Too much DNA on gel
-Preparation of

LB medium and Agar

1% and 0,7% agarose

SD-Ura medium (+Trp)

SD-Ura medium for plates (add 100 ml of 20% Galactose and 10% Raffinose -> already prepared)

40% Glucose

-Miniprep of CDS-IAAM and CDS-IAAH
concentrations

CDS-IAAH
clone 1	315 ng/µl	clone 3	247,8 ng/µl
clone 7	298,1 ng/µl	clone 5	356,7 ng/µl
clone 8	340,2 ng/µl	clone 6	386, 4 ng/µl
clone 9	437,3 ng/µl	clone 7	287,4 ng/µl
clone 10	389,6 ng/µl	clone 8	232,6 ng/µl

-test digest of CDS-IAAM and CDS-IAAH
-gel picture: bands correct! expected lengths:

CDS-IAAM: 900 bp + 6500 bp

CDS-IAAH: 1100 bp + 6050 bp

Thursday, 10/05/17

-Colony PCR of Clones of masterplate from 10/02/2017
->clone 9 and 16 did not grow
-1 single clone of each clone segment (1-8 and 10-15) was picked and resuspended in 50 µL 0.02 M NaOH
-the cell suspensions incubated for 15 min at 37°C
-In parallel the Mastermix for the Colony PCR for 16 samples was prepared:

component	volume (µl)
H20	174.4 µL
IG_X_Q25	12.8 µL
IG_X_Q102	12.8 µL
MAstermix ALLin	160 µL

--> 22.5 µL were added to 14 PCR tubes (Labeled like HcP # (#=1-9,10-15))
-2.5 µL of the in NaOH resuspended yeast clones were added to the mastermix in the corresponding tubes
-Start of PCR program:

step	temp (°C)	time	cicles
Initial Denaturation	98°C	1 min
Denaturation	98°C	15 s	35x
Annealing	55°C	15 s
Extension	72°C	30 s
final extension	72°C	5 min

-2 µL of PCR product + 2 µL 6x Loading Dye + 8 µL h2O
--> load on a 0.7 % agarose gel
--> run for 60 min at 120 V
--> there are no bands visible!
-Repetition of gradient PCR with Ddx4-IAAM-Ddx4-IAAH
-6 different samples:
-->3x Gibson Assembly from 2.10 1:100 diluted
-->3x GibsonAssembly from 2.10 1:1000 diluted
-this time higher dilutions are used, because gel from 4.10 showed white "smear" --> Assumely the amount of DNA which was used for PCR was too high -Again 3 different annealing temperatures (58, 59 and 61 °C) are used
-For each dilution factor a Mastermix (for 3 samples) was prepared:

component	volume
H20	75 µL
IG_X_103	7.5 µL
IG_X_Q104	7.5 µL
Q5 Mastermix	57 µL

-in each tube 49 µL Mastermix and 1 µL templare are added
-final 6 samples:

1) dF 100 58°C

2) dF 100 59°C

3) dF100 61°C

4) dF1000 58°C

5) dF1000 59°C

6) dF1000 61°C

--> Start PCR Program

step	temp (°C)	time	cycles
Initial Denaturation	98°C	30 s
Denaturation	98°C	10 s	35x
Annealing	58/59/61°C	30 s
Extension	72°C	171 s
final extension	72°C	5 min

--> 50 µL PCR product + 10 µL 6x Loading Dye
--> Load on a 0.7% agarose gel
--> the estimated size of the PCR product is ~5700 bp
--> No bands with this size are visible
-->in next step we will do a Gibson Assembly to clone Ddx4-IAH and Ddx4-IAAM in the pYES-CT_d_PmeI_HindIII backbone in one reaction
-Gibson Assembly to clone Ddx4-IAAH and Ddx4-IAAM in the pYES-CT_d_PmeI_HindIII backbone
-the IDT fragments of Ddx4-IAAH and Ddx4-IAAM are used
-Assembly composition:

Ddx4-IAAH (c=50 ng/µL)	0.97 µL	--> 48.68 ng	-->97.9 ng
Ddx4-IAAM (c=50 ng/µL)	0.98 µL	--> 49.22 ng	-->97.9 ng
pYES-CT_d_PmeI_HindIII	1.8 µL	--> 50 ng
H2O	6.25 µL
Gibson Assembly Mix	10 µL

-Incubation for 15 min at 50°C
-->Transformation in chemical competent NEB10beta cells
--> cells were plated on LB-Amp plates
-SD-Ura plates
--> SD-Ura plates are finished

Friday, 10/06/17

-repeat colony PCR from 5.10.17 taking clones from the master plate from 2.10.17
-this time trying 2 different cell lysis methods:
1.) clones were resuspended in 50µl 0,02M NaOH and for 15 minutes incubated at 37°C (N)
2.) clones were resuspended in 20µl 0,25% SDS, 10sec vortexing, 3 min incubated at 90°C and centrifuged for 30sec, supernatant took for Colony-PCR (S)
-From each clone segment 1-8 and 10-15, 1 clone per lysis method was taken
-PCR Mastermix: 289,5µl H2O; 24µl IC_X_Q102; 24µl IG_X_Q25; 337,5µl Q5 master mix (because taq mastermix was nearly empty)
-each PCR-tube: 22,5µl mastermix + 2,5µl resuspended clone
-PCR program: initial dneaturation: 95°C 1min, (denaturation 95°C 15s, annealing 55°C 15s, extension 72°C 30s) x 35, final extension 72°C 5min
-2µl PCR product + 8µl water + 2µl 6xloading dye --> 0,7% agarose gel (60min, 140V)
-Gel picture:
-band at 900-1000 bp could indicate positive clones
-band should be at 992bp
-ligation of Ddx4-YFP in pSB1C3 after dephosphorylation of the pSB1C3 backbone:
-dephosphorylation:
-9µl pSB1C3 (12,5ng/µl) + 2µl 10xCutSmart buffer + 1µl Quick CIP + 8µl H2O
-Incubation at 37°C for 10 minutes
-heat inactivation for 2min at 80°C
-ligation: 8,9µl pSB1C3 (dephosphorylated, 50ng) + 4,4µl Ddx4-YFP digested wih EcoRI and PstI (132,4ng) + 1µl ligase + 2µl ligase buffer + 3,7µl H2O
->over night at 16°C

Saturday, 10/07/17

-heat inactivation (65°C, 10 min) of Ligation mix (Ddx4-YFP + pSB1C3, 06.10.17)
-Transformation of 5 µl Ligation mix into DH5a (NEB)
-plated on cap plates

->incubation: at 28°C until monday

Monday, 10/09/17

-Miniprep of clones N3 and N12 of plate from 6.10.
-concentrations:

N3: 13 ng/uL

N12: 5,1 ng/ul

N6 was streaked again beaucse it only showed one colony
5 uL of the miniprep was transformed into 25 uL competent Dh5alpha cells and plated on Amp
Colony PCR of DDx4-YFP in pSB1C3 (from 7.10.)
Mastermix for 12 reactions:

component	volume (°C)
Q5 Mastermix	60
IG_X_Q17	6
IG_X_Q18	6
H2O	48

-10 uL per reaction -10 clones were picked and one was for control -PCR program: 35 cycles

temp (°C)	time
98	2min
98	10s
52	20s
72	17s
72	2min

-added loading dye and ran gel
-all colonies negative
-picked 17 clones again and all negative too (no bands at 1.7 kb)
-clone 21 shows a wrong band and was incubated in 5ml culture with Amp, incubation over night

Tuesday, 10/10/17

-Miniprep of 4 ml of the culture from October 9th ( DDx4-YFP in pSB1C3, Amp, clone 21)
-> elution in 50 µl water
->final concentration: 406,0 ng/µl
-Test digest of this clone:

component	volume
NEBuffer 2.1	1 µl
EcoRI-HF	0,5 µl
PstI	0,5 µl
water	7 µl
Ddx4-YFP in pSB1C3 (200 ng/µl) -> used 300 ng	1 µl

-200ng/µl dilution has been done before
-incubation for 60 min at 37°
-test gel (0,7%, ~40 min): whole digest (10 µl) + 2 µl LD
-result: two bands around 2 kb
-expected lengths: 1750 bp and 2000 bp
-clone 21 sent for sequencing with Q17 (ID 33FE47) and Q18 (ID 33FE48), because it might be correct anyway (gel was generally -behaving weirdly; went very slow, solvent front was somehow curved)
_____________________________
-New approach to get Ddx4-YFP into pSB1C3: take backbone that has already taken an insert and cut insert out (just in case something is wrong with our backbone)
______________________________
-Digest of IG_C_P_CAP_TAC1 with EcoRI and PstI to gain backbone (pSB1C3)

component	volume
NEBuffer 2.1	1 µl
EcoRI-HF	0,5 µl
PstI	0,5 µl
water	3 µl
IG_C_P_CAP_TAC1 (265,7 ng/µl -> 1,3285 µg)	5 µl

-incubation for 90 min at 37° -gel clean up (0,7% agarose) ->final concentration: c(pSB1C3_d_E+P) = 17,9 ng/µl
-dephosphorylation of backbone

component	volume
pSB1C3 d E+P (17,9 ng/µl)	23,5 µl
CutSmart	4 µl
Quick CIP (phosphorylase)	2 µl
H2O	10,5 µl
IG_C_P_CAP_TAC1 (265,7 ng/µl -> 1,3285 µg)	5 µl

-incubation for 10 min at 37 °
-heat inactivation for 2 min at 80°
-in parallel: digest of Ddx4-YFP (44 ng/µl) with Gibson overhangs (compare tto p.17 in Low copy lab book)

component	volume
NEBuffer 2.1	1 µl
EcoRI-HF	0,5 µl
PstI	0,5 µl
water	1,2 µl
Ddx4-YFP (44 ng/µl) with Gibson overhangs	6,8 µl -> 300 ng

-heat inactivation for 20 min at 80°
-Ligation of Ddx4-YFP_d_E+P and pSB1C3_d_E+P (10,52 ng/µl)

component	volume
T4 Ligase buffer	2 µl
T4 Ligase	1 µl
pSB1C3_d_E+P -> 50 ng	4,75 µl
Ddx4-YFP_d_E+P -> 132.4 ng	4,4 µl
water	7,85 µl

-incubation over night at 16°
-Dephosphorylation of digested pSB4A5 from october 9th

component	volume
pSB4A5_d_E+P	48 µl
CutSmart	6 µl
Quick CIP (phosphorylase)	3 µl
water	3 µl

->incubation for 10 min at 37°
->heat inactivation for 2 min at 80°
-Preparation of miniprep cultures from transformation from october 9th (pYES2/CT-Ddx4-IAA)
-> several clones from N3 and N12 (40x 5 ml cultures in test tubes because there were no more 100 ml flasks)

Wednesday, 10/11/17

-Miniprep according to protocoll: the 2 cultures per clone were combined and cleaned up on one column
->elution with water (50 µl/ 100 µl)
-concentrations:
-Ddx4 IAA in pYES (3)

clone	concentrations (ng/µl)
1.1	124,5
1.2	110,5
1.3	96,4
1.4	123,0
1.5	208,8

-Ddx4 IAA in pYES (N12)

clone	concentrations (ng/µl)
1.6	108,3
1.6	88,8
1.8	104,6
1.9	71,2
1.10	91,2
2.1	103,5
2.2	98,5
2.3	80,0
2.6	224,2
2.7	199,1
2.8	75,4
2.9	154,5
2.10	109,9

-test digest of all

component	volume
NEBuffer 2.1	1 µl
EcoRI-HF	0,5 µl
PstI	0,5 µl
water	3 µl
Ddx4-IAA in pYES2/CT	5 µl

->incubation for 1h at 37° -> Cancelled
-new test digest for all
-batch for 22 aliquots

component	volume
CutSmart	22 µl
BamHI	4,4 µl
water	149,6 µl
(Miniprep	44 µl)
	149,6
total	220 µl

8 µl master mix + 2 µl miniprep per clone

incubation for 15 min at 37° (time saver enzyme!)

Test gel: 0,7%, 140V, 40+23 min

expected lengths: 3283 bp, 1404 bp, 6730 bp

->result: definitely not (only two bands for most of them, some have three but wrong sizes)

Ligation over night (16°) of IAA fusion (42,6 ng/µl) with digested pSB4A5 (E+P) (23,28 ng/µl)

component	volume
T4 Ligase buffer	2 µl
T4 Ligase	1 µl
pSB4A5 E+P -> 50 ng	2,15 µl
IAA fusion	3,01 µl
water	11,84 µl

-Transformation of ligation from 10.10.17 (Ddx4-YFP in pSB1C3) in competent DH5alpha (plated 200µl and rest on Amp-plates) -Evaluation of sequencing results of:

IAAH-CDS: Sequencing result is positive

IAAM-CDS: Sequencing result is positive

Ddx4-YFP (clone 11): Sequencing result is positive

-Evaluation of BamHI test digestion of Ddx4-IAAs in pYES2CT (digestionnjust 15 min -> next time 60 min): bands at ~1,5kb and ~6-8kb -> prediction: IAAM is missing (might be cut out during recombination in yeast at homologues regions)
-IAA-Ddx4-Fusion:
-> IDT-fragments amplified again with GXL DNA Polymerase
-> IAA-Ddx4 fusion (from 5.10.) again amplified with GXL DNA Polymerase
Master mix for 2 reactions:

29µl H2O; 10µl 5xGXL buffer

4µl dNTP mixture

2µl Primer 1

2µl Primer 2

1µl GXL DNA Polymerase

-> 24µl master mix + 1µl template
-IAAH-Ddx4: Primer 1: IG_X_Q105; Primer 2: IG_X_Q106 -> template concentration a) 1:100 b) 1:1000 diluted
-IAAM-Ddx4: Primer 1: IG_X_Q103; Primer 2: IG_X_Q_104 ->template concentration a) 1:100 b) 1:1000 diluted
-IAA-Ddx4-fusion: Primer 1: IG_X_Q103; Primer 2: IG_X_Q106 ->template concentration a) 1:10 b) 1:100 diluted
-PCR program: denaturation: 98°C 10s; Annealing 55°C 15s; Elongation 68°C 5min42s

add 5µl 6xloading dye to PCR sample

load 3µl PCR sample (+loading dye) in small pockets to check sizes of bands

load residue to pockets for gel clean up

both on one big gel

run gel for 45min at 140V

-> no gel clean-up possible becasue very strange gel run, no bands but a lot of smear
-IAAH-CDS insertion into IAAM-CDS-pYES2CT
-digestion of IAAM-CDS-pYES22-CT to open vector
-->3µl 10x CutSmart
-->1,5µl NaeI
-->10,4µl IAAM-CDS pYES2-CT (3µg)
-->15,1µl H2O
->digest for 4h at 37°C
-dephosphorylation:
30µl digested IAAM-CDS pYES2CT
1,3µl QuickCIP (no addition of CutSmart buffer necessary because the digestion was already in CutSmart buffer, if it would be in another buffer 3,5µl 10xCutSmart buffer would need to be added)
->incubation at 37°C for 30 min (10min are enough)
->put on 0,7% agarose gel, run gel for 45 min at 140V
-Gel clean up -> 30,4ng/µl
-PCR amplification of pYES-IAAH with IG_X_Q118/119 -> 50µl reaction:

25µl Q5_Mastermix

2,5µl IG_X_Q118

2,5µl IG_X_Q119

19µl H2O 1µl template (pYES-CDS-IAAH) ->1ng/µl
-PCR-program (35 cycles)

temp (°C)	time
98	30s
98	10s
66	30s
72	3min 39s
72	2min

add 10µl loading dye

load on 0,7% agarose gel

3µl -> for checking correct size

57µl -> for gel clean up

-> overloaded, bands move differently

-gel clean up -> 37,3ng/µl
-Gibbson-Assembly IAAH + IAAM-pYES (digested and dephosphorylated):

50ng IAAM-pYES 1,6µl

29,5ng IAAH casette 0,8µl

10µl mastermix

7,6µl water

-> 15min at 50°C
-transformation with 5µl Gibbson-Assembly in DH5alpha

->amp plates 200µl + rest

-Yeast miniprep of N6 (pYES-Ddx4-IAAs)
-transformation of plasmid N6 of the miniprep in JM109 (E.coli) cells using 5µl plasmid
-on amp plates 200µl and rest

Friday, 10/13/17

0,7% agarose gel -> 52min 120V:

1-6: 25ng
1: Ddx4-IAAM (IDT) 2893bp 50ng/µl -> 0,5µl + 9,5µl water + 2µl loading dye
2: Ddx4-IAAH (IDT) 2871bp 50ng/µl -> 0,5µl + 9,5µl water + 2µl loading dye
3: IAAH-cassette 2196bp 37,3ng/µl -> 0,7µl
4: pYES2-CT-CDS-IAAM dNaeI 7441bp 30,4ng/µl -> 0,8µl
5: P4 linearised with Q11/12 7309bp 4,7ng/µl -> 5,3µl
6: LacI-dCas9 (with overhangs to P4) 5680bp 114,1ng/µl -> 0,2µl
7: Gibson Assembly of P6 (from 12.10.17) 12983bp -> 3µl
concentration of IDT fragments measured at nanodrop:
Ddx4-IAAH:35,2ng/µl
Ddx4-IAAM: 44,9ng/µl
Colony-PCR of IAA-cassttes (CDS) in pYES2-CT - Transfromation of 12.10.17
-2x10 clones
-first 10 clones were negative, probably because of wrong elongation temperature (take care for that now, we have another taq-mastermix now, with a lower elongation temperature!!!)
mastermix for 12 reactions:

120µl 2xTaq-mastermix; 4,8µl IG_X_Q120; 4,8µl IG_X_Q121; 110,4µl H2O

20µl per tube (10tubes) pick clone from plates transfer to masterplate and then resuspend cells in mastermix

PCR program:

initial denaturation: 95°C 30s

denaturation: 95°C 20s

annealing: 52°C 30s

elongation: 68°C 77s

final elongation: 68°C 5min

->load an 1% agarose gel

->clone 12,17,18,19,20 show bands at 1281bp, where they should be
-from this clones liquid cultures (5ml) were prepared and incubated over night at 37°C

troubleshooting:
-concentration of IDT fragments shpuld be 50ng/µl but is not (see gel picture, there is nothing to see from the IDT fragments)
-->5µl of the IDT fragments were load to a 0,7% agarose gel, shpuld be 250ng, but was around 25ng -> 10x smaller than it should be
-fragments must be cleaned up from the gel

11,5µl fragments with loading dye were load to 1% agarose gel -> 30min 120V

bands were cut out -> tomorrow gel clean up

-transformation of pYES2-CT-IAAM_d_NaeI (12.10.17) 30,4ng/µl -> 3,3µl and IAAH-cassette (12.10.17) 37,3ng/µ -> 2,7µl in competent yeast-cells; 100ng each
-protocol Zymo research (incubation at 30°C for 1h20min)
-to SD-Ura plates plated all (because else is was always to little growing)
incubation for 3 days -> monday colony-PCR

Saturday, 10/14/17

Ddx4-IAAM -> 6,4 ng/µl
Ddx4-IAAH -> 5,0 ng/µl
-PCR for amplification of of IAAM/IAAH

	IAAM 1/IAAM 10	IAAH 1/IAAH 10
component	volume	volume
Q5	12,5 µl	12,5 µl
X_Q103	1,25 µl	-
X_Q104	1,25	-
X_Q105	-	1,25 µl
X_Q106	-	1,25 µl
template	2 µl / 2 µl (1:10)	2 µl/ 2 µl (1:10)
water	6,125 µl	6,125 µl

Program:

temp (°C)	time	cycles
98	30s
98	10s	30x
63,1° (for IAAM) and 68,2° (for IAAH)	20s
72	70s
72	2min

Test gel: IAAM 1, IAAH 1 and IAAH 10 positive (but somehow slurred)
-New PCR because of wrong gradient
-new approach for IAAM and IAAH

approach	volume (dilution)	temp °C)
1	1 µl (1:1)	55°
2	1 µl (1:1)	60°
3	1 µl (1:1)
4	1 µl (1:10)
5	1 µl (1:10)	60°
6	1 µl (1:10)	2 step

-> 24 µl Maser mix per reaction
-Mastermix for IAAM:

component	volume
buffer (PrimeStar)	35 µl
IG_X_Q103	7 µl
IG_X_Q104	7 µl
dNTP	17 µl
water	108,5 µl

Mastermix for IAAH:

component	volume
buffer (PrimeStar)	35 µl
IG_X_Q105	7 µl
IG_X_Q106	7 µl
dNTP	17 µl
water	108,5 µl

-program for tubes 1,2,4 and 5 (no 2 step):

temp (°C)	time
98°	10s
gradient (55° - 60°)	15s
68°	3 min

30 cycles
Program for tubes 3 and 6 (2 step):

temp (°C)	time
98°	10s
68°	3min

-Miniprep of the IAA cassettes in pYES2/CT

elution in 50 µl water

-final concentrations:

clone 12: 243,1 ng/µl

clone 17: 316,0 ng/µl

cloone 18: 271.9 ng/µl

-> clone 19&20 ngative according to test digest

Test digest:

component	volume
CutSmart (10x)	1 µl
HindIII-HF	0,2 µl
Miniprep (clones 12,17,18,19,20)	2 µl
water	6,8 µl

-test gel (0,7%, 40 min)

expected bands at 7537 bp, 1151 bp, 699 bp

clones 12,17 and 18 possibly positive

-Glycerol stocks (1:1) of 12 and 17
-prepared sequencing of 17 with X_Q107 (33FE49), X_Q108 (33FE50), X_Q122 (33FE51), X_Q123 (33FE52), X_Q124 (33FE53)
-Transformation of the IAA cassettes in pYES2/CT into competent yeast cells (clones 12 and 17)

50 µl competent cells + 100 ng DNA (c(12): 243,1 ng/µl & c(17): 316,0 ng/µl)

V(12) = (100 ng / (243,1 ng/µl) = 0,411 µl

V(17) = 0,316 µl

+500 µl EZ3 solution

-After inoculation (30° -> 45 min) plated 50 µl and 150 µl on SD-URA with Glucose (No Galactose, Raffinose, Tryptophane)
-wrapped with Parafilm

Monday, 10/16/17

-test gel of gradient PCR from Saturday 14.10 (120 V. 25 min)
-result: several bands

-> preparative gel with about 25 µl + 9,5 µl (we thought that the PCR from 14.10 has been 50 µl aliquote)

gelextraction (Ddx4-IAAH: 2839 bp and Ddx4-IAAM 2871 bp) and clean up

c(Ddx4-IAAH) = 112,1 ng/µl

c(Ddx4-IAAM) = 67,6 ng/µl

transformation of Ddx4-IAAH, Ddx4-IAAM and pYES2/CT (digested with HindIII-HF and PmeI (27.09.) in competent yeast cells following yeast protocoll
-digest of pYES2/CT from 27.09 repeated
pYES 25.08.17; 162,4ng/µl

component	volume
pYES2/CT (2 ng)	12,3 µl
Cutsmart	3 µl
water	12,7 µl
HindIII-HF	1 µl
PmeI	1 µl
total	30 µl

Incubation: 1,5h at 37°C

digestion on 0,7% agarose gel -> gel extraction

gel clean up: c(pYES2/CT digested with HindIII-HF and PmeI) = 20,3 ng/µl

(on gel also 50 ng Ddx4-IAAH und DDx4-IAAM)

-> band Ddx4-IAAH (2839 bp) a little bit smaller than band Ddx4-IAAM (2871 bp)
-amplification-PCR of Ddx4-IAAM-Ddx4-IAAH-fusion
-with three different templates and six different temperatures
mastermix (20x):

component	volume
5x Q5 Reaction buffer	100 µl
10 mM dNTPs	10 µl
10 mM IG_X_Q103	25 µl
10 mM IG_X_Q106	25 µl
Q5 Polymerase	5 µl
GC Enhancer	100 µl
ddH2O	251 µl
total	480 µl

-24 µl each pro tube + 1 µl template
-templates:

- undiluted Gibson-Assembly*

- dilution 1:10 Gibson-Assembly*

- dilution 1:100 Gibson-Assembly*

*Gibson-Assembly:

component	volume
Ddx4-IAAH (112,1 ng/µl ≙ 100 ng)	0,9 µl
Ddx4-IAAM (67,6 ng/µl ≙100 ng)	1,5 µl
ddH2O	7,6µl
HiFi-Mastermix	10 µl

Incubation 20 min at 50 °C

PCR-program:

initial denaturation: 98 °C 30s

denaturation: 98 °C 10s

annealing: 63°C 30 s

extension: 72 °C 171 s

final extension: 72 °C 2 min

35 cycles

Gibson-Assembly with 3 fragments:

component	volume
pYES2/CT (digested with HindIII-HF and PmeI): 28,2 ng/µl -> 50 ng	1,8 µl
Ddx4-IAAH (112,1 ng/µ -> 49,22 ng)	0,4 µl
Ddx4-IAAM (67,6 ng/µ -> 48,68 ng)	0,7 µl
ddH2O	7,1 µl
HiFi-Mastermix	10 µl
total	20 µl

incubation 20 min at 50 °C

transformation in DH5alpha

-yeast transformation from Saturaday 14.10 pYES-IAA-cassette clone #12 and #17 on master plate

Tuesday, 10/17/17

preparative gel:

0,7%, of the "amplification-PCR of Ddx4-IAAM-Ddx4-IAAH-fusion"

25µl + 5µl loading Dye

120V, 30min

classification of the wells:

U58/58(1:10)/58(1:100)/U59/Ladder/59(1:10)/59(1:100)/U61/61(1:10)/61(1:100)

U65/65(1:10)/65(1:100)/67(1:10)/Ladder/U67/67(1:100)/U68/68(1:10)/68(1:100)

cut out 3 lines: 58(1:100); U59; 61(1:100)

->very greasy

-gel clean up
->final concentration: 16,6 ng/µl
-Testgel to check the size
->3µl template (=50ng/146ng/µl) + 7µl H2O + 2µl loading Dye
-colony PCR of the transformation (16.10.17) of Gibson-Assembly pYES2-CT+Ddx4-IAAM+Ddx4-IAAH
Mastermix for 10 approaches:

component	volume
H2O	80µl
IG_X_Q25	10µl
IG_X_Q102	10µl
TaqMastermix	100µl

PCR Program:

temp (°C)	time	cycles
95°C	30sec
95°C	20sec	53x
32°C	30sec
68°C	1min
68°C	5min

-Testgel (0,7%) after clean up (Ddx4-IAAM-Ddx4-IAAH)
result: should be 50ng and 5680bp --> definitely lower concentration!
-again: plate out wildtype (YPH500 on YPDA-plate)
-repeated colony PCR
-colony PCR of the transformation (16.10.17) of Gibson-Assembly pYES2-CT+Ddx4-IAAM+Ddx4-IAAH
Mastermix for 10 approaches:

component	volume
H2O	80µl
IG_X_Q120	10µl
IG_X_Q108	10µl
TaqMastermix	100µl

PCR program:

temp (°C)	time	cycles
95°C	30sec
95°C	20sec	53x
51°C	30sec
68°C	2min20sec
68°C	5min

-Testgel (0,7%)

->10 positiv clones = 10 minipreps

->inoculate 10 minipreps (10ml LB+Agar with clone (1 to 10))

->over night at 37°C

Wednesday, 10/18/17

-Miniprep (pYES2-CT+Ddx4-IAAM+Ddx4-IAAH) from yesterdays 17.10
-Digestion:

component	volume
Miniprep	2 µl
BamHI	0,2 µl
Cutsmart	1 µl
ddH2O	6,8 µl
total	10 µl

incubation for 1 h at 37 °C
gel (0,7 % agarose gel 30 min 120v)

-> all clones three bands, except clone 2

-IAA-cassettes for iGEM HQ from 14.10: 12, 17, 18 1:1 and 1:10 dilution each (= templates)
PCR master mix:

component	volume
5x Q5 reaction buffer	30 µl
10 mM dNTPs	3 µl
10 µM IG_X_Q127	7,5 µl
10 µM IG_X_Q128	7,5 µl
Q5 Polymerase	1,5 µl
GC Enhancer	30 µl
ddH2O	64,5 µl

-> 24 µl Mastermix + 1 µl template
PCR program:

temp (°C)	time	cycles
98 °C	30sec
98 °C	10 sec	35x
61 °C	30sec
72 °C	2min25sec
78 °C	2 min

-> 2 µl PCR product + 8 µ ddH2O + 2 µl loading dye

on 1 % agarose gel

-result:
->only clone 17 (1 ng/µl) showed a band

band should be 4.820 bp, is probably too small

-PCR clean up from clone 17 (1 ng/µl)
->c (clone 17) = 84 ng/µl
-digestion from IAA-cassette with BioBrick prefix suffix

component	volume
EcoRI-HF	0,5 µl
PstI	0,5 µl
NEB 2.1	1 µl
template (83 ng/µl -> 40 ng)	4,8 µl
ddH2O	3,2 µl

incubation 1h t 37 °C

20 min 80 °C inactivation

Thursday, 10/19/17

-repeated PCR of IAA-cassette for iGEM-HQ with GXL Polymerase

12 reactions: 60µl 5xGXL Buffer, 24µl 10mM dNTPs, 6µl IG_X_Q127, 6µl IG_X_Q128, 6µl GXL DNA Polymerase, 186µl Wasser

--> 188µl Mastermix

--> 24µl Mastermic per tube

+ 1µl template each

-1ng/µl and 0,1ng/µl respectively of clone 12,17 and 18 as template (2x, because different annealing temperatures)
-> 1x55°C Annealing temperature
-> 1x68°C Annealing temperature

temp (°C)	time	cycles
98 °C	10sec
98 °C	10 sec	30x
55 °C/68°C	15sec
68 °C	4min50sec
78 °C	2 min

-load on 0,7% agarose gel

Results

-IAA cassette to small, perhaps just 1 fragment inside

-Colony-PCR of CDS-IAAs clone 12 from yeast (master plate of monday 16.10.17)

7 reactions: 70µl Tag-Mastermix + 2,8µl IG_X_Q120 + 2,8µl IG_X_Q121 + 46,9µl water

clone in 50µl 0,02M NaOH each resuspended and for 15min at 37°C incubated

-> 2,5µl of resudoended culture to 17,5µl mastermix

-clone 1 to 5 were streaked again

-> 1 plate divided in thirds and plated clone 1,2,3

-> 2 plate bisected and plated clone 4 and 5

-PCR-Programm: 95°C 30s, (95°C 30s, 53°C 60s, 68°C 1min17s)x35, 68°C 5min
-yeast trafo with IAA-cassettes clone 12 Miniprep -> 50ng/µl dilution and 2µl used for transformation

Friday, 10/20/17

-Yeast-transformation with Ddx4-IAA in pYES
-prepared 50 ng/µL solution of Ddx4-IAA in pYES
-transformed 2 µL in competent yeast cells
-plated cellls on SD-Ura plates and incubated at 30°C
Sequencing:

pYES-IAA-cassette = C12: template tube = 20,56µl DNA + 29,4µl H2O (final concentration 100ng/µl), Primer 10µM = IG_X_Q107,102,120,122,108,111,123

pYES-Ddx4-IAAs = C6: template tube = 13,55ng/µl DNA + 41045µl H2O (final concentration 100ng/µl), Primer 10µM = IG_X_Q122,102,107,104,109,120,117,26

-Coloy-PCRnCDS-IAAs clone 12:
-repeated with other colonies and the same colonies from yesterday that were stored at 4°C (the resusension of the cells in 0,02M NaOH), because gel was negative, because maybe the primers were interchanged
-new clones resuspended in 0,02M NaOH and incubated for 15min at 37°C

17,5µl mastermix (140µl Taq; 5,6µl IG_X_Q120; IG_X_Q121; 93,8µl Water (mastermix for 14 reactions)) +2,5µl resuspended clone

-PCR program: 95°C 30s, (95°C 30s, 53°C 60s, 68°C 1min17s)x35cycle, 68°C 5min
-> load to 1% agarose gel

->negative

-Ddx4-IAAs-PCR to add BioBrick Prefox and Suffix
mastermix:

component	volume
5xGXL Buffer	20 µl
10mM dNTPs	8 µl
IG_X_Q125	2 µl
IG_X_Q126	2 µl
GXL DNA Polymerase	2 µl
water	62µl

->24µl mastermix per tube + 1µl template respectively
-template: 1ng/µl clone 6, clone 9 two times for two different annealing temperatures 68°C and 55°C

temp (°C)	time	cycles
98 °C	10sec
98 °C	10 sec	30x
55 °C/68°C	15sec
68 °C	6min31sec

-load to 1% agarose gel (test gel: 2µl PCR reaction + 8µl H2O + 2µl loeading dye) -> 120V 30min
--> PCR clean up (clone 6 and clone 9 separated, but the both annealing temperatures together to one column)

-> clone 6: 65.8 ng/µl

-> clone 9: 56.3 ng/µl

-Restriction of Ddx4-IAAs with BioBrick sites with EcoRI and PstI

component	volume
template (Ddx4-IAAs clone 6/8)	16 µl
EcoRI-HF	1 µl
PstI	1 µl
NEB buffer 2.1	2 µl

--> incubate at 37°C for 1h

--> Heat inactivation for 20 min at 80 °C

-Ligation of digested Ddx4-IAAs with BioBrick sites in digested pSB1C3
-Composition of ligation sample:

component	volume
template (Ddx4-IAAs clone 6/8)	x µl
T4 Ligase	1 µl
T4 Ligase buffer	1 µl
vector (pSB1C3)	2 µl
water	to 20 µl

-Needed volumes of template "x":
-Ddx4-IAAs clone 6: 7.4 µL
-Ddx4-IAAs clone 8: 8.7 µL
-->incubation at 16°C over night

Saturday, 10/21/17

-Transformations from pYES-Ddx4-IAAs cl.6 (18.10.) and pYES-IAA-cassettes cl.12 (19.10.) into yeast didnt look good -> seemed like nothing will grow -> to be sure we put them back in the 30°C incubator
-Repeated transformation of pYES-Ddx4-IAAs cl.6 (405.7 ng/µl, 18.10.) and pYES-IAA-cassettes cl.12 (243.1 ng/µl, 14.10.) into yeast
-diluted each plasmid to 100 ng/µl
-used 1 µl (100 ng) of dilution for transformation
-Incubation at 30°C for 100 min
-plated on SD-Ura + glucose plates (150 µl + rest)
-Transformation of 5 µl ligation mix of Ddx4-IAA-cassettes (cl. 6 / 9) + pSB1C3 into DH5a
-plated on CAP plates
-incubation over night at 37°C

Sunday, 10/22/17

-Colony PCR of Ddx4-IAAs in pYES2CT (using masterplate from the 19.10)
-Pick 8 clones from the Masterplate and resuspend them in 25 µL 0.02 M NaOH
--> Incubate at 37°C for 15 min
Mastermix for 10.8 samples (there was a mistake in the calculations)

component	volume
108.2 µL	Quick Load Taq Mastermix
4.8 µL	IG_X_108
4.8 µL	IG_X_120
82.2 µL	H2O
water	10 µl

-aliquote 17.5 µL Mastermix in the tubes and add 2.5 µL resuspended clon (+1 control without clone resuspension)
-Start PCR Program:

step	temp (C°)	time
Initial Denaturation	95 °C	30s
Denaturation	95°C	20s
Annealing	51°C	30s
Elongation	68°C	2min20s
Final Elongation	68°C	5min

--> Load on a 0.7 % Agarose gel
--> Colony PCR was negative
--> repitition with new calculated volumes:
Mastermix for 9 samples:

component	volume
Quick Load Taq Mastermix	90 µL
IG_X_108	3.6 µL
IG_X_120	3.6 µL
H2O	60.3 µL

-Aliquote 17.5 µL Mastermix to the tubes and add 2.5 µL ressupended clone (8 samples + 1control)
--> Start PCR Program (same as above)
-->PCR was again negative
Preparation of Masterplates:
-->2 SD-Ura plates were divided in 8 segments
-->8 clone of Ddx4-IAA in pYES and IAA-cassettes in pYES were picked and streaked out on the segmented plates, respectively Incubation at 30 °C
Ddx4-YFP in pYES
-->a SD-Ura (+Glc) plate is divided into 2 halves
-->clone 2 and 7 were picked from plate frome 25.9 and plated on the SD-Ura (+Glc) plate
-->incubation at 30 °C

Monday, 10/23/17

Sent for sequencing: Ddx-IAA-fusions in pYES (Miniprep of october 10th)

B454112: clone 7 (+ Q117)

B454111: clone 8 (+ Q117)

B454110: clone 9 (+Q117)

Trafo of clones sent for sequencing: pYES2/CT Ddx4 IAA fusion into competent yast cells
-->clone 7: V= 100 ng/452,1 ng*µl = 0,203 µl
--> not possible to pipet -> dilution 1:10 -> 2,03 µl miniprep of clone 7

clone 8: (1:10) V= 4,94 µl

clone 9: (1:10) V= 3,63 µl

-incubation for 1h 15 min at 30°
-platet 150 µl and rest
-SD media again black! (That was the third try!)
-From now on: Neither Trp, Glucose, Raffinose or Galactose will be autoclaved (only steril filtrated)
-Did a test if black slides come from contamination: culture with 5 ml medium in shaker (37°)
-measured OD(600) (23.10., 10:30Uhr) -> OD= 0,071 (blank - empty cuvette)
-Please repeat measurement tomorrow!! (We kept one bottle with the black medium)
-Prepared Yeast media
-Induction medium for clones

component	volume
SD - Ura medium	600 ml
10 g/l Trp	600 ml -> 4 g/l final concentration
20% Galactose	150 ml
10% Raffinose (penta hydrate)	150 ml

for 1,5 L SD-Ura+Trp+Gal+Raf

2. Repressive medium for clones

component	volume
SD - Ura medium	600 ml
10 g/l Trp	600 ml -> 4 g/l final concentration
20% Glucose	300 ml

for 1,5 L SD-Ura+Trp+Glu

3. Induction medium for wild type

component	volume
SD + Ura medium	600 ml
10 g/l Trp	600 ml -> 4 g/l final concentration
20% Galactose	150 ml
10% Raffinose (penta hydrate)	150 ml

for 1,5 L SD+Ura+Trp+Gal+Raf

4. repressive medium for wild type

component	volume
SD + Ura medium	600 ml
10 g/l Trp	600 ml -> 4 g/l final concentration
20% Glucose	300 ml

for 1,5 L SD+Ura+Trp+Glu

Tuesday, 10/24/17

Pre-Culture for GC-MC-measurement:

pYES-Ddx4-YFP clone 2 with 10ml SD-Ura+Glucose Media

pYES-Ddx4-YFP clone 7 with 10ml SD-Ura+Glucose Media

pYES-IAA-cassettes clone 1 with 10ml SD-Ura+Glucose Media

pYES-IAA-cassettes clone 2 with 10ml SD-Ura+Glucose Media

pYES-IAA-cassettes clone 3 with 10ml SD-Ura+Glucose Media

pYES-IAA-cassettes clone 5 with 10ml SD-Ura+Glucose Media

YPH500 I with 10ml YPDA Media

YPH500 II with 10ml YPDA Media

-scratched out pYES-IAA-cassettes clone 1, 2, 3, 5 to inoculate tomorrow Pre-Cultures for Yeast-Minipreps
-Colony PCR of pYES-Ddx4-IAA
-8 clones resuspend in 25µl 0,02M NaOH and incubate 15min at 37°C
-Mastermix for 9 Reactions

component	volume
Taq master mix	90 µl
IG_X_Q108	3,6 µl
IG_X_Q120	3,6 µl
water	60,3 µl

-->17,5µl MasterMix+2,5µl Template
PCR Program:

step	temp (°C)	time
Initial Denaturation	95 °C	30s
Denaturation	95°C	20s
Annealing	51°C	30s
Elongation	68°C	2min20s
Final Elongation	68°C	5min

Gel: 0,7%, 120V, 30min
--> Result: all clones negativ
Colony PCR of pYES-IAA-cassettes
-->5 clones resuspend in 25µl 0,02M NaOH and incubate 15min at 37°C
Mastermix for 6 Reactions

component	volume
Quick Load Taq Mastermix	60 µL
IG_X_108	2,4 µL
IG_X_120	2,4 µL
water	40,3 µL

-17,5µl MasterMix+2,5µl Template
PCR Program (30 cycle)

98°C 10s

68°C/55°C 15s

68°C 6min 31s

-Gel: 0,7%, 120V, 30min
-all clones negativ

Wednesday, 10/25/17

-Minipreps of yesterday's precultures for the gc ms measurements (Yeast miniprep!)
-> elution in 10 µl water, no measurement of concentration
-Transformation of those (clone 1,2,3,5) into competent JM109
-Yesterday`s preculttures have grown in wrong medium (medium contained Uracil). Therefore new cultures were inoculated
-Just in case the new don't grow: main cultures were made out of the "wrong" precultures (x ml were taken from every preculture, centrifuged and then transfered into new medium)
-See protocoll for gc ms on Box.up!

Thursday, 10/26/17

Yeast colonies for GC-MS:

pYES IAA cassette clone 1,2,3 and 5

pYES Ddx4-YFP clone 2 and 7

pYES Ddx4-IAAs clone 8.1, 8.2, 9.1, 9.2, 9.3

-each in SD - Ura + Trp + Glu (green label on the flask) and SD - Ura + Trp + Gal + Raf (red)
YPH 500 I and II:
-each in SD + Ura + Trp + Glu (blue) and SD + Ura + Trp + Gal + Raf (yellow)
-inoculated at 16:30 -> grow till tomorrow 8:30

Friday, 10/27/17

-centrifuged and extract (MPI) cultures for GC-MS-calculation (inoculated on 25.10.17)
-Measurement on wednesday, November 1st

Team:Potsdam/notebook/LLPS

IGEM Potsdam