Team:KIT-Kyoto/Notebook/LabNote/GFPMLF8

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22nd, August

Members

Yoshimura, Nakagawa, Matsunami

We have transformed E. coli DH5 alpha with the plasmid BBa_E0240.

Results

The transformed bacterial colonies were obtained.

23rd, August

Members

Yoshimura, Nakagawa

1. Pre-culture of bacteria transformed with BBa_E0240.

2. Design of primers to construct GFP plasmid without ATG from the plasmid BBa_E0240.

Results

1. We have succeeded the preculture.

2. The designed primers are shown below.

F: 3' TTGAATTCTTTCTAGATTCGTAAAGGAGAAGAA

　　　　　 　　 EcoRⅠ　　　XbaⅠ

Tmvalue:67.75°C　　33 base

R: 5' AAACTGCAGAAAACTAGTTTTTTATTATTTGTATAG

　　　　　 　 　　 PstⅠ　　　SpeⅠ

Tmvalue:67.66°C　　36 base

24th, August

Members

Yoshimura、Nakagawa

After isolation of plasmid DNA BBa_E0240 by the alkaline-lysis method, DNA samples were digested by the following restriction enzymes at 37 ℃ for 30 min.

ddH2O	2 µl
BBa_E0240	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

After restriction enzyme digestion, samples were electrophoresed in 1% agarose gel at 100 V for 30min.

Results

DNA bands in the agarose gel.

You can see DNA band at around 800bp.

25th, August

Members

Yoshimura、Nakagawa

PCR reaction was carried out by using primers designed on 23rd. The reaction conditions are summarized below.

PCR reaction 10 µM GFP Primer F 1.5 µl 10 µM GFP Primer R 1.5 µl BBa_E0240 1 µl 10 x PCR Buffer for KOD Plus 5 µl dNTPs 4 µl MgSO4 4 µl ddH2O 32 µl KOD Plus 1 µl   total 50 µl

Cycle条件 Pre-Denature 95°C 30sec   Denature 95°C 30sec 30 Cycle Anneling 50°C 1min Extension 68°C 1min End 4°C keep

Agarose gel electrophoresis was carried out to detect the PCR products.

Then the PCR products were digested with the following restriction enzymes (at 37°C for 16 hours).

ddH2O	2 µl
BBa_E0240	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

Results

DNA bands in the agarose gel.

You can see DNA band at around 800bp.

26th, August

Member

Nakagawa

1.Extraction of DNA from the agarose gel.

2.Transformation of E. coli DH5 alpha with the plasmid pSB1C3.

Results

1. Picture of agarose gel after cutting out of the portion containing the DNA fragment.

Final concentration of the isolated DNA was 510 ng/µl.

2. Although small colonies were observed, we decided to transform the bacteria again just in case.

27th, August

Member

Yoshimura

Transformation of E. coli DH5 alpha with the plasmid pSB1C3

Results

The transformed bacterial colonies were obtained.

29th, August

Member

Yoshimura, Nakagawa

Pre-culture of pSB1C3(6 sample)

Results

We have succeeded to grow bacteria from three samples out of six.

30th, August

Member

Nakagawa

1.Pre-culture of pSB1C3(6 sample)

2.Design of primers to construct GFP plasmid without ATG from the plasmid BBa_E0240.

Results

1.We have succeeded the preculture.

2.The designed primers are shown below.

F: 3' TTGAATTCTTTTCTAGATTTCGTAAAGGAGAAGAA

　　　　　 　　 EcoRⅠ　 　　XbaⅠ

Tm value :68.8゜C　　35 base

31st, August

Member

Nakagawa

After isolation of pSB1C3 by the alkaline-lysis methodfollowed by phenol-chloroform treatment, DNA was digested with the following restriction enzymes (30 min at 37℃).

ddH2O	2 µl
BBa_E0240	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

After restriction enzyme digestion, DNA samples were electrophoresed at 100 V for 30min.

Results

Image of the agarose gel.