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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.
22rd, 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
- F: 3' TTGAATTCTTTCTAGATTCGTAAAGGAGAAGAA
EcoRⅠ XbaⅠ
- Tmvalue:67.75°C 33 base
- Tmvalue:67.75°C 33 base
- R: 5' AAACTGCAGAAAACTAGTTTTTTATTATTTGTATAG
- 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.
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- 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.
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- 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
- 中川
- 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
- F: 3' TTGAATTCTTTTCTAGATTTCGTAAAGGAGAAGAA
EcoRⅠ XbaⅠ
- Tm value :68.8゜C 35 base
- 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.
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