Team:KIT-Kyoto/Notebook/LabNoteJ/DIAP2-MALT8
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8月8日
Member
- Matsunami, Yokoigawa
- To amplify API2-MALT1 cDNA and DIAP2cDNA, PCR reactions were carried out by using the following primers and under the following conditions.
- API2-MALT1
- F primer:GCCGCTCGAGAACATAGTAGAAAACAGCAT
- Tm value 57.8°C
- R primer:GCCGGCTAGCTCATTTTTCAGAAATTCTGA
- Tm value 56.5°C
- amplicon size 3131 bp
- F primer:GCCGCTCGAGAACATAGTAGAAAACAGCAT
- DIAP2
- F primer:GCTTCTCGAGACGGAGCTGGGCATGGAGCT
- Tm value 69°C
- R primer:GCCGTCTAGATCACGAAAGGAACGTGCGCA
- Tm value 66.4°C
- amplicon size 1514 bp
- F primer:GCTTCTCGAGACGGAGCTGGGCATGGAGCT
- 1.API2-MALT1
PCR reaction 10 µM Primer F 0.4 µl 10 µM Primer R 0.4 µl Template DNA 1 µl 10 x Ex Taq Buffer 2 µl Takara Ex Taq 0.1 µl 2.0 mM dNTPs 2 µl ddH2O 14.1 µl total 20 µl Cycle Pre-Denature 94°C 2min Denature 94°C 30sec 37 Cycle Anneling 51.5°C 30sec Extension 72°C 3min20sec +Extension 72°C 10min End 4°C keep
- 2.DIAP2
PCR reaction 10 µM Primer F 0.4 µl 10 µM Primer R 0.4 µl Template DNA 1 µl 10 x Ex Taq Buffer 2 µl Takara Ex Taq 0.1 µl 2.0 mM dNTPs 2 µl ddH2O 14.1 µl total 20 µl Cycle Pre-Denature 94°C 2min Denature 94°C 30sec 37 Cycle Anneling 56.9°C 30sec Extension 72°C 1min40sec +Extension 72°C 10min End 4°C keep
8月9日
Member
- Matsunami, Yokoigawa
- 1. We carried out agarose gel electrophoresis to detect the PCR products.
- 2. We transformed E. coli XL-1 blue with the plasmid pUAST-flag.
Results
- 1. Image of the agarose gel.
- Lane 1;size marker(1 kbp ladder)
- Lane 2;the amplified API2-MALT1 cDNA fragment
- Lane 3;the amplified DIAP2 cDNA fragment
- Sizes of the fragments were different from the expected sizes.
- 2. The transformed bacterial colonies were detected.
10th, August
Member
- Matsunami
- 1. I have repeated the PCR reactions to amplify API2-MALT1 cDNA and DIAP2 cDNA under the same conditions as those carried out on 8th, August.
- 2. I picked up the colonies and grew them in liquid culture.
Results
- 2. I have successfully grown the transformed bacteria.
11th, August
Member
- Matsunami
- I have isolated the plasmid DNAs (pUAST flag ) by alkaline-lysis method and electrophoresed them in agarose gel.
Results
- Image of the agarose gel.
- Lane 1; size marker (1 Kbp ladder)
- Lanes 2~9;pUAST-flag
- Lane 10;The authentic pUAST-flag vector
- Lane 11;the amplified API2-MALT1 cDNA fragment
- Lane 12;the amplified DIAP2 cDNA fragment
12th, August
Member
- Matsunami, Yokoigawa
- 1. Amounts of the isolated plasmid DNA samples were quantified by measuring the absorbance at OD260.
- 2. We grew the transformed bacteria (25 ml x 2) and the plasmid pUAST-flag DNA was isolated by Midi-prep (Invitrogen).
Results
- 1. The absorbance at OD260 of two independent plasmid DNA samples are shown in Table 1.
- Table 1 (OD260)
- sample 1
0.189 0.208 0.192 0.190 0.191 - Average value was 0.194.
- Concentration of DNA was 0.970 µg/µl.
- sample 2
0.282 0.276 0.278 0.269 0.269 - Average value was 0.275.
- Concentration of DNA was 1.37 µg/µl.
2. Image of the agarose gel.
- Lane 1: DNA size marker (1Kb ladder)
- Lane 6: sample 1
- Lane 7: sample 2
- Lane 8: The authentic pUAST-flag vector
- DNAs from samples 1 and 2 migrated to the same positions as the pUAST-flag.
15th, August
Member
- Matsunami, Yokoigawa
- We have tried different Annealing conditions to amplify API2-MALT1 cDNA and DIAP2 cDNA. The other conditions for the PCR reactions were same as those carried out on 8th, August.
- API2-MALT1
Anneling 58.9°C
- DIAP2
Anneling 53°C
- PCR products were applied to the agarose gel electrophoresis.
Results
- Image of the agarose gel.
- Lane 1;DNA size marker(1 Kbp ladder)
- Lane 2;API2-MALT1 cDNA
- Lane 3;DIAP2 cDNA
- Size of the amplified fragments for API2-MALT1was different from the expected size and multiple DNA fragments were detected for the DIAP2.
16th, August
Member
- Matsunami
- Again different annealing conditions were tested.
- API2-MALT1
Anneling 53°C
- DIAP2
Anneling 58.9°C
- PCR products were applied to the agarose gel electrophoresis.
Results
- Image of the agarose gel.
- Lane 1;DNA size marker(1 Kbp ladder)
- Lanes 2 and 3;API2-MALT1 cDNA
- Lane 4;DIAP2 cDNA
- In lane 3, the DNA fragment with the expected size (3 kb) with the additional 2kb fragment was detected for API2-MALT1. Multiple :DNA fragments were still detected for the DIAP2.
17th, August
Member
- Matsunami, Yokoigawa
- Again different annealing conditions were tested.
- API2-MALT1
Anneling 53.5°C
- DIAP2
Anneling 50.5°C
- PCR products were applied to the agarose gel electrophoresis.
Results
- Image of the agarose gel.
- Lane 1;DNA size marker(1 Kbp ladder)
- Lanes 2 and 3;API2-MALT1 cDNA
- Lane 4;DIAP2 cDNA
- Size of the amplified fragments for API2-MALT1was different from the expected size and amplified DNA fragments were barely detectable for the DIAP2.
8月23日
Member
- 松浪
- API2-MALT1、DIAP2について、8月8日と同じ条件でAnnelingを以下のように変更してPCRを行った。
- API2-MALT1
Anneling 53°C
- DIAP2
Anneling 50.5°C
- PCR産物が増幅していることを確認するため、電気泳動を行った。
Results
- 泳動後の写真
左から順に1レーン;マーカー(1 Kbp)、2レーン;API2-MALT1、3レーン;API2-MALT1、4レーン;DIAP2、5レーン;DIAP2
API2-MALT1についてDNAポリメラーゼを変更したが、期待されるバンドは見られなかった。
8月24日
Member
- 松浪
- API2-MALT1について10、100倍希釈後、以下の条件でPCRを行った。
PCR条件 10 µM Primer F 0.4 µl 10 µM Primer R 0.4 µl Template DNA 1 µl 10 x Ex Taq Buffer 2 µl Takara Ex Taq 0.1 µl 2.0 mM dNTPs 2 µl ddH2O 14.1 µl total 20 µl Cycle条件 Pre-Denature 94°C 2min Denature 94°C 30sec 37 Cycle Anneling 51.5°C 30sec Extension 72°C 3min20sec +Extension 72°C 10min End 4°C keep
- PCR産物が増幅していることを確認するため、電気泳動を行った。
Results
- 泳動後の写真
左から順に2レーン;マーカー、3~6レーン;API2-MALT1(10倍希釈)、8~11レーン;API2-MALT1(100倍希釈)
バンドは何も見られなかった。
8月25日
Member
- 松浪
- API2-MALT1、DIAP2について、8月8日と同じ条件でAnnelingを以下のように変更してPCRを行った。
- API2-MALT1
Anneling 51.5°C
- DIAP2
Anneling 50.5°C
- PCR産物が増幅していることを確認するため、電気泳動を行った。
Results
- 泳動後の写真
左から順に1レーン;マーカー(1 Kbp)、2・3レーン;API2-MALT1、4・5レーン;DIAP2
API2-MALT1に関してバンドは見られなかった。DIAP2については予想される位置にバンドが見られた。
8月29日
Member
- 武田、横井川
- 25日に確認したDIAP2をフェノールクロロホルム処理し、
- pUAST-flag vectorとともに、下表に従って制限酵素処理を行った。(37°C、overnight)
- DIAP2
DIAP2 PCR産物 in ddH2O 44 µl 10 x H Buffer 5 µl XhoⅠ 1 µl total 50 µl pUAST-flag vector pUAST-flag vector(500 ng/µl) 10 µl ddH2O 34 µl 10 x H Buffer 5 µl XhoⅠ 1 µl total 50 µl
37°Cで20時間静置した。
8月30日
Member
- 武田、横井川
- 25日に確認したDIAP2をフェノールクロロホルム処理し、
- pUAST-flag vectorとともに、下表に従って制限酵素処理を行った。(37°C、overnight)
DIAP2 PCR産物 in ddH2O 44 µl 10 x M Buffer 5 µl XbaⅠ 1 µl total 50 µl pUAST-flag vector pUAST-flag vector(500 ng/µl) 10 μl ddH2O 34 µl 10 x H Buffer 5 µl XhoⅠ 1 µl total 50 µl
37°Cで20時間静置した 。
8月30日
Member
- 武田、横井川
- QIAquick Gel Extraction Kitを使用してゲルからDIAP2のDNAとpUAST-flag vectorを抽出した。
Results
- ゲル切り出し後の写真
- pUAST-flag vectorは精製でき、濃度は45.0375 ng/µlであった。
- DIAP2はゲル抽出でバンドが見られなかった。