Team:KIT-Kyoto/Notebook/LabNote/DIAP2-MALT9

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1st, September

Member

Takeda

Again different annealing conditions were tested.

PCR reaction 10 µM Primer F 1.5 µl 10 µM Primer R 1.5 µl Template DNA 1 µl 10 x PCR Buffer 5 µl dNTPs 5 µl MgSO4 2 µl or 4 µl ddH2O 33 µl or 31 µl KOD+ polymelase 1 µl   total 50 µl

Cycle Pre-Denature 94°C 2min   Denature 94°C 15sec 35 Cyle Anneling 50.5°C 30sec Extension 68°C 100sec End 4°C keep

PCR products were applied to the agarose gel electrophoresis.

Results

Image of the agarose gel.

5th, September

Member

Yokoigawa, Takeda

The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.

DIAP2

PCR reaction in ddH2O	44 μl
10 x H Buffer	5 μl
XhoⅠ	1 μl
	total 50 μl

6th, September

Member

Yokoigawa, Takeda

The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the XbaI for 20 hours at 37°C.

DIAP2

PCR reaction in ddH2O	44 μl
10 x M Buffer	5 μl
XbaⅠ	1 μl
	total 50 μl

7th, September

Member

Yokoigawa, Takeda

The restriction enzyme-digested PCR fragments and pUAST-flag DNA were extracted from the agarose gel by[http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx QIAquick Gel Extraction Kit].

Results

PCR products for DIAP2 Was not successfully recovered from the agarose gel.

12th, September

Member

Matsunami, Yokoigawa

To amplify DIAP2 cDNA, PCR reactions were carried out by using the following primers and under the following conditions.

F primer GCTTCTCGATTGACGGAGCTGGGCATGGAGCT

Tm value　　　　62 °C

R primer GCCGTCTAGATCACGAAAGGAACGTGCGCA

Tm value　　　　66.4°C

amplicon size　　1514 bp

PCR reaction 10 µM Primer F 1.5 µl 10 µM Primer R 1.5 µl Template DNA 1 µl 10 x PCR Buffer 5 µl dNTPs 5 µl MgSO4 2 µl ddH2O 33 µl KOD+ polymelase 1 µl   total 50 µl

Cycle Pre-Denature 94°C 2min   Denature 94°C 15sec 35 Cycle Anneling 50.5°C 30sec Extension 68°C 1min40sec End 4°C keep

Again different annealing conditions were tested.

Annealing 50.5°C

13th, September

Member

Matsunami, Yokoigawa

PCR products on 12th September 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；DIAP2 cDNA

Amplified DNA fragments were barely detectable for the DIAP2.

14th, September

Members

Matsunami,Yokoigawa

To amplify API2-MALT1 cDNA and, PCR reactions were carried out by using the following primers and under the following conditions.

F primer GCCGCTCGAGACATAGTAGAAAACAGCAT

Tm value　　　　57.7 °C

R primer GCCGGCTAGCTCATTTTTCAGAAATTCTGA

Tm value　　　　56.5 °C

amplicon size　　約3131 bp

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 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

No PCR product was detected for API2-MALT1.

15th, September

Members

Matsunami,Yokoigawa

To amplify API2-MALT1 cDNA and DIAP2cDNA, PCR reactions were carried out by using under the following conditions.

API2-MALT1

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 53°C 30sec Extension 72°C 3min20sec +Extension 72°C 10min   End 4°C keep

DIAP2

PCR条件 10 µM Primer F 1.5 µl 10 µM Primer R 1.5 µl Template DNA 1 µl 10 x PCR Buffer for KOD Plus 5 µl dNTPs 5 µl MgSO4 2 µl ddH2O 33 µl KOD Plus 1 µl   total 50 µl

Cycle条件 Pre-Denature 94°C 2min   Denature 94°C 15sec 35 Cycle Anneling 50.5°C 30sec Extension 68°C 1min 40sec End 4°C keep

PCR products were applied to the agarose gel electrophoresis.

The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.

DIAP2

PCR産物 in ddH2O	44 µl
10 x H Buffer	5 µl
XhoⅠ	1 µl
	total 50 µl

Results

Image of the agarose gel.

Lane 1；DNA size marker（1 Kbp ladder）

Lanes 2~5；DIAP2 cDNA

Lane 6~8；API2-MALT1 cDNA

９月１６日

Member

松浪、横井川

DIAP2のPCR産物を、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

９月１７日

Member

松浪、横井川

制限酵素処理を行ったDIAP2のプラスミドを確認するため、電気泳動を行った。

Results

泳動後の写真

マーカー（1 Kbp）のバンドのみが見られた。制限酵素処理を行ったDIAP2のサンプルはすべてバンドが見られなかった。

９月１８日

Member

松浪

コンピテントセル（DH5α）を作成した。

９月１９日

Member

松浪

コンピテントセル（DH5α）を作成した。

９月２１日

Member

松浪

1.コンピテントセル（DH5α）を作成した。

2.DIAP2について、以下の条件でPCRを行った。

PCR条件 10 µM Primer F 1.5 µl 10 µM Primer R 1.5 µl Template DNA 1 µl 10 x PCR Buffer 5 µl dNTPs 5 µl MgSO4 2 µl ddH2O 33 µl KOD+ polymelase 1 µl   total 50 µl

Cycle条件 Pre-Denature 94°C 2min   Denature 94°C 15sec 35 Cycle Anneling 50.5°C 30sec Extension 68°C 1kb/min 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。

DIAP2のPCR産物を、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 H Buffer	5 µl
XhoⅠ	1 µl
	total 50 µl

Results

1.ODを測定したが、OD＝0.9を超えてしまった。

泳動後の写真

左から順に1レーン；マーカー（1 Kbp）、2～7レーン；DIAP2

全てのサンプルでPCRが成功した。

９月２２日

Member

不明★

DIAP2のPCR産物を、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

９月２３日

Member

松浪

制限酵素処理を行ったDIAP2のプラスミドを確認するため、電気泳動を行った。

Results

泳動後の写真

マーカー（1 Kbp）のみバンドが見られ、制限酵素処理を行ったDIAP2のサンプルはすべてバンドが見られなった。

９月２５日

Member

松浪

コンピテントセル(DH5α)を作成した。

９月２７日

Member

松浪

コンピテントセル(DH5α)を作成した。