Team:KIT-Kyoto/Notebook-week3p

September 7th

1-1-33 and 2-1-23 PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA

DNA template	0.25uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH2O	34.15uL
Total	50uL

Reaction conditions

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
58℃	2min30sec	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)
14℃	∞

1-1-34 and 2-1-24 PCR products were applied to the agarose gel electrophoresis
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below



Results: no clearly amplified bands were detected.

Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6

2-1-25 SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL


From these results, concentration of the pSB1C3 DNA was estimated to be 10ng/uL.

GAL4
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker 2uL, DNA 0.1uL, 1kbmarker 1uL



However no GAL DNA was detected. We lost the sample somewhere by some mistakes.

September 8th

1-1-35 and 2-1-26
PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA

DNA template	1uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH2O	33.4uL
Total	50uL

Reaction conditions

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
58℃	2min30sec	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)
14℃	∞

1-1-36 and 2-1-27 PCRproducts applied to the agarose gel electrophoresis
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each


Only the PCR products for pSB1C3 was detectable.

1-1-37 and 2-1-28
We repeated PCR for UAS and GAL4DNA by changing the annealing temperature
（-1 indicates 55℃、-2 indicates 58℃）

DNA template	1uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH2O	33.4uL
Total	50uL

Reaction conditions

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
55℃(-1) or 58℃(-2)	2min30sec	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)
14℃	∞

September 9th

1-1-38 and 2-1-29
PCRproducts were electrophoreses.
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each


We finally succeeded the amplification of the UAS fragments.

1-1-39 Purification of pSB1C3 and UAS-1-PCR products
PCR products were purified by High Pure PCR Product Kit

1-1-40
The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction

UAS	40uL
4 buffer(NEB)	5uL
EcoRⅠ-HF(NEB)	0.5uL
SpeⅠ(NEB	0.5uL
100×BSA	0.5uL
dH2O	3.5uL
Total	50uL

1-1-41
The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit



2-1-30 PCR amplification of GAL4

DNA template	1uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH2O	33.4uL
Total	50uL

Reaction conditions

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
58℃	2min30sec	30 cycle
68℃	2min10sec	30 cycle
68℃	2min10sec
14℃	∞

2-1-31
PCR products were applied to the agarose gel electrophoresis as shown below.


1-1-42
pSB1C3 and UAS fragments were ligated in the following reactions.

pSB1C3 (EcoRⅠ and SpeⅠ digested)	1uL
UAS (EcoRⅠ and SpeⅠ digested)	1uL
dH2O	3uL
Ligation high	2.5uL
Total	7.5uL

The following reaction were carried out as a negative control.

pSB1C3 (EcoRⅠ and SpeⅠ digested)	1uL
dH2O	4uL
Ligation high	2.5uL
Total	7.5uL

1-1-43
Ligation products(1-1-42) were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(＋) plate and incubated at 37℃ for 16 hours.

September 10th

At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.

1-1-44 Single colony isolation of ligationproducts
Ligation product prepared on 9/9 (UAS and pSB1C3) gave us some colonies and negative control sample gave us no colony. We therefore picked up four independent colonies and cultured them in 2.5mL LB Chloramphenicol (＋) medium at 37℃ for 16 hours.

2-1-32 Digestion of HS promoter fragment and Act5C promoter with XbaⅠ and BamHⅠ
DNA fragments carrying HS promoter (prepared on 9/5) and those carrying Act5C promoter (prepared on 9/3) were digested with XbaⅠ and BamHⅠ.

HS or Act5c	40uL
4 buffer(NEB)	5uL
XbaⅠ-HF(NEB)	0.5uL
BamHⅠ(NEB　)	0.5uL
100×BSA	0.5uL
dH2O	3.5uL
Total	50uL

Digested fragments were purified from the gel by QIA quick Gel Extraction Kit.

Photo of agarose gel


2-1-33 PCR amplification of GAL4 fragments
We tried PCR under four different conditions described below.

	G-1 and G-2	G-3 and G-4
DNA template	1uL	4uL
10× KOD plus buffer	5uL	5uL
2mM dNTPs	5uL	5uL
25mM MgSO4	1.6uL	1.6uL
10P 5’ primer	1uL	1uL
10P 3’ primer	1uL	1uL
KOD plus	1uL	1uL
dH2O	33.4uL	31.4uL
Total	50uL	50uL

Reaction conditions

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
57℃(G-1 and G-3) or 59℃(G-2 and G-4)	2min30sec	30 cycle
68℃	2min10sec	30 cycle
68℃	2min10sec
14℃	∞

2-1-34 PCR products were applied to agarose gel electrophoresis
From left to right: 10uL each G-1, G-2, G-3, G-4

Photo of agarose gel


We found that GAL4 sequence was properly amplified under G-1 and G-2conditions.

2-1-35
PCR products were purified by High Pure PCR Product Kit.

1-2-1 Digestion of pSB1C3 DNA with EcoRⅠ and SpeⅠ
PCR-amplified pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ for 2 hours at 37℃.

pSB1C3(PCR)	40uL
4 buffer(NEB)	5uL
EcoRⅠ-HF(NEB)	0.5uL
SpeⅠ(NEB)	0.5uL
2100×BSA	0.5uL
dH2O	3.5uL
Total	50uL

1-2-2 Digestion of EcoRI-digested UAS fragment by BglⅡ
BglⅡ-digestion was carried out for 2 hours at 37℃ under conditions described below.

UAS(EcoRⅠ-digested)	13uL
3 buffer(NEB)	2uL
BglⅡ(NEB)	0.5uL
dH2O	2.5uL
Total	20uL

1-2-3 Purification of restriction enzyme-digested pSB1C3 DNA and UAS fragment
restriction enzyme-digested pSB1C3 DNA and UAS fragment (prepared on 9/10) were purified by QIA quick Gel Extraction Kit.

Photo of agarose gel


2-1-36
PCR-amplified GAL4 fragments were digested with XbaⅠ and SpeⅠ under conditions described below.

GAL4	20uL
4 buffer(NEB)	4uL
XbaⅠ(NEB)	0.7uL
SpeⅠ(NEB)	0.7uL
100×BSA	0.4uL
dH2O	14.6uL
Total	40uL

The digested DNA was applied to agarose gel electrophoresis as shown below.

Photo of agarose gel


Results: XbaⅠ-digestion of GAL4 fragment gave two DNA fragments, indicating that GAL4 sequence contains XbaⅠ site.

2-2-1 Digestion of GAL4 sequence with BglⅡ and SpeⅠ
Digestion was carried out st 37℃ for 1hour under the conditions described below.

GAL4	40uL
3 buffer(NEB)	5uL
BglⅡ(NEB)	0.5uL
dH2O	4.5uL
Total	50uL

The digested DNA was purified by High Pure PCR Product Kit was further digested with Spe I at 37℃ for 1hour.

GAL4(Bgl Ⅱ cut)	40uL
4 buffer(NEB)	5uL
SpeⅠ	0.5uL
100×BSA	0.5uL
dH2O	4uL
Total	50uL

The digested fragments were purified by QIA quick Gel Extraction Kit.

Photo of agarose gel


1-2-4 and 2-2-2
Insertion of GAL4 fragments and HS promoter or Act5C promoter-enhancer and UASfragment and EGFP sequence or LacZ sequence into the pSB1C3DNA

Ligation reactions were carried out at 16℃ for 1hour under conditions described below.

pSB1C3 (PCRproducts)(double digested with EcoRⅠ and SpeⅠ)	1uL
UAS (double digested with EcoRⅠ and BglⅡ)	2uL
EGFP or LacZ	2uL
Ligation high	5uL
Total	10uL

pSB1C3 (vector DNA) (double digested with XbaⅠ and SpeⅠ)	1uL
GAL4 (double digested with BglⅡ and SpeⅠ)	2uL
HS or Act5c (double digested with XbaⅠ and BamHⅠ)	2uL
Ligation high	5uL
Total	10uL

1-2-5 and 2-2-3
Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(＋) plate and cultured at 37℃for 16 hours.

September 11th

1-2-6 Single colony isolation
Single colonies were isolated from the plate prepared on 9/11 and cultured in 2.5mL LB Chloramphenicol(＋) medium at 37℃ for 16 hours.

1-1-45 Isolation of the candidate pSB1C3-UAS DNA
The candidate pSB1C3-UAS DNA was purified by QIA prep Spin Miniprep Kit.

1-1-46
The purified candudate pSB1C3-UAS was digested with BglII for 2 hours.

pSB1C3-UAS-1, -2, -3, -4	5uL
3buffer(NEB)	2uL
BglⅡ	0.5uL
dH2O	12.5uL
Total	20uL

The digested samples were applied to agarose gel electrophoresis as shown below. Left to right: pSB1C3-1 uncut, pSB1C3 cut, -2 uncut, -2 cut, -3 uncut, -3 cut, -4 uncut, -4 cut

Photo of agarose gel


Results: All DNAs examined had a single BglII site, indicating that the pSB1C3-UAS DNA was successfully constructed.

The BglII-digested pSB1C3-UAS DNA was purified from the gel by QIA quick Gel Extraction Kit.

2-2-3 Digestion of pSB1C3 (PCR) with XbaⅠ and SpeⅠ
PCR-amplified pSB1C3 DNA was double digested with XbaⅠ and SpeⅠ at 37℃ for 2 hours.

GAL4	40uL
4 buffer(NEB)	5uL
XbaⅠ(NEB)	0.5uL
SpeⅠ(NEB)	0.5uL
CIP	0.5uL
100×BSA	0.5uL
dH2O	3uL
Total	40uL

The digested samples were purified by QIA quick Gel Extraction Kit

Photo of agarose gel


2-2-4 Insertion of GAL4, HS promoter or Act5C promoter-enhancer into the pSB1C3DNA
Ligation reactions were carried out under conditions described below at 16℃ for 1hour .

pSB1C3 (vector) (digested with XbaⅠ and SpeⅠ)	1uL
GAL4 (digestion with BglⅡ and SpeⅠ)	2.5uL
HS promoter or Act5C promoter-enhancer (digested with XbaⅠ and BamHⅠ)	2.5uL
Ligation high	6uL
Total	12uL

pSB1C3 (PCR products) (digested with XbaⅠ and SpeⅠ)	2uL
GAL4 (digested with BglⅡ and SpeⅠ)	2.5uL
HS promoter or Act5C promoter-enhancer (XbaⅠ and BamHⅠ)	1.5uL
Ligation high	6uL
Total	12uL

2-2-5
Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(＋) plate and cultured at 37℃for 16 hours.

September 12th

1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs
We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11).

1-1-47 Digestion of pSB1C3-UAS DNA by Spe1
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.

Composition

pSB1C3-UAS-1, -2	40uL
3buffer(NEB)	5uL
SpeⅠ	1uL
100×BSA	0.5uL
dH2O	3.5uL
Total	50uL

The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.

Photo of Agarose gel.


1-1-48 and 2-2-6 Ligation
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.

Ligation reactions are listed below.

pSB1C3-UAS (double digested with BglII and SpeⅠ)	1uL
EGFP fragments or LacZ fragments	2uL
EGFP or LacZ	2uL
Ligation high	2.5uL
dH2O	2uL
Total	7.5uL

We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.

Composition

pSB1C3 (PCR) (double digested with XbaⅠand SpeI)	2uL
GAL4(double digested with BglⅡ and SpeⅠ)	2uL
HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ)	2uL
Ligation high	6uL
Total	12uL

1-1-49 and 2-2-7 Transformation of E. coli by Ligation products
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.