Team:KIT-Kyoto/Notebook/LabNoteJ/DIAP2-MALT8

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

DIAP2

F primer:GCTTCTCGAGACGGAGCTGGGCATGGAGCT

Tm value　69°C

R primer:GCCGTCTAGATCACGAAAGGAACGTGCGCA

Tm value　66.4°C

amplicon size　　1514 bp

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

[http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx 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。