Team:KIT-Kyoto/Notebook-week1p

First

The first number 1 indicates experiments about UAS.
The first number 2 indicates experiments about GAL4.
Second number 1 indicates making two experiments at the same time when we carried out ligation.
Second number 2 indicates making three experiments at the same time when we carried out ligation.
The parts we are going to submit are these seven, pSB1C3-UAS, pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, pSB1C3-UAS-TNFAIP3,pSB1C3-GAL4, pSB1C3-HS-GAL4, and pSB1C3-Act5c-GAL4.

August 23rd

1-1-1 and 2-1-1 Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(＋) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(＋) plate.

August 24th

1-1-2 and 2-1-2
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.

August 25th

1-1-3 and 2-1-3 Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.

1-1-4 Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt
Restriction enzyme digestion was carried out in the following reaction

Total DNA amount	500ng	1ug
pEGFP-C	3uL	6uL
H buffer(TOYOBO)	5uL	5uL
BamHⅠ(TOYOBO)	1uL	1uL
BglⅡ(TOYOBO)	1uL	1uL
100×BSA	0.5uL	0.5uL
dH2O	39.5uL	36.5uL
Total	50uL	50uL

1-1-5 Agarose gel electrophoresis of the digested DNA
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.

Photograph of the agarose gel


1-1-6 Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.

DNA sample	23uL
4 buffer(NEB)	5uL
EcoRⅠ-HF(NEB)	1uL
SpeⅠ(NEB)	1.5uL
100×BSA	0.5uL
dH2O	19uL
Total	50uL

1-1-7 PCR amplification of the UAS region and pSB1C3 DNA
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.

DNA template	0.5uL
10× KOD plus buffer	10uL
2mM dNTPs	10uL
25mM MgSO4	3.2uL
10P 5’ primer	3uL
10P 3’ primer	3uL
KOD plus	2uL
dH2O	68.3uL
Total	100uL

Reaction conditions

Temperature	Time	Circle
95℃	2min
95℃	15sec	25 cycle
58℃	30min(UAS) or 2min10sec(pSB1C3)	25 cycle
68℃	2min30sec	25 cycle
68℃	2min30sec
14℃	∞

August 26th

1-1-8 Purification of the digested pSB1C3
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.

Photo of the agarose gel

August 27th

1-1-9 Removing the multi-cloning site of the pEGFP-C2
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.

pEGFP-C2(cut)	1uL
Ligation high	2.5uL
dH2O	5uL
Total	7.5uL

1-1-10
The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(＋) plate and cultured at 37℃ for 16 hours.

1-1-11 Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.

Photo of the agarose gel


Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below.

Photo of the agarose gel

August 28th

1-1-12 Single colony isolation from the E. coli transformed with pEGFP-C2 DNA
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(＋) medium at 37℃ for 16 hours.

2-1-4 Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(＋) plate for 16 hours at 37℃.

August 29th

2-1-5 Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(＋) medium for 16 hours at 37℃.

1-1-13 Purification of pEGFP-C2 DNA
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.

1-1-14 Cut check of the pEGFP-C2 DNA
The purified pEGFP-C2(1-1-13) was digested with XhoⅠ and PstⅠ in the following reactions.

pEGFP-C2-1, -2, -3, -4	1uL
10×H buffer(TOYOBO)	0.5uL
XhoⅠ	0.1uL
PstⅠ	0.1uL
dH2O	3.3uL
Total	5uL

The digested samples were applied to the agarose gel electrophoresis.as shown below.

Photo of the agarose gel


Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.

1-1-15 PCR amplification of the DNA fragments containing UAS and EGFP
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.

DNA template	0.5uL
10× KOD plus buffer	10uL
2mM dNTPs	10uL
25mM MgSO4	3.2uL
10P 5’ primer	3uL
10P 3’ primer	3uL
KOD plus	2uL
dH2O	68.3uL
Total	100uL

Reaction conditions

Temperature	Time	Cycle
95℃	2min
95℃	15sec	25 cycle
58℃	30sec	25 cycle
68℃	30sec(UAS) or 1min(EGFP)	25 cycle
68℃	30sec(UAS) or 1min(EGFP)
14℃	∞

Agarose gel electrophoresis of the PCR products


Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS.