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

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8th, August

Member

Matsunami, Yokoigawa


To amplify API2-MALT1 cDNA and DIAP2 cDNA, 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 F0.4 µl
10 µM Primer R0.4 µl
Template DNA1 µl
10 x Ex Taq Buffer2 µl
Takara Ex Taq0.1 µl
2.0 mM dNTPs2 µl
ddH2O14.1 µl
 total 20 µl
Cycle
Pre-Denature94°C2min 
Denature94°C30sec37 Cycle
Anneling51.5°C30sec
Extension72°C3min20sec
+Extension72°C10min 
End4°Ckeep 


2.DIAP2
PCR reaction
10 µM Primer F0.4 µl
10 µM Primer R0.4 µl
Template DNA1 µl
10 x Ex Taq Buffer2 µl
Takara Ex Taq0.1 µl
2.0 mM dNTPs2 µl
ddH2O14.1 µl
 total 20 µl
Cycle
Pre-Denature94°C2min 
Denature94°C30sec37 Cycle
Anneling56.9°C30sec
Extension72°C1min40sec
+Extension72°C10min 
End4°Ckeep 


9th, August

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
0.1890.2080.1920.1900.191
Average value was 0.194.
Concentration of DNA was 0.970 µg/µl.


sample 2
0.2820.2760.2780.2690.269
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
Anneling58.9°C
DIAP2
Anneling53°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-MALT1 was 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
Anneling53°C
DIAP2
Anneling58.9°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
Anneling53.5°C
DIAP2
Anneling50.5°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-MALT1 was different from the expected size and amplified DNA fragments were barely detectable for the DIAP2.


23rd, August

Member

Matsunami


Again different annealing conditions were tested.
API2-MALT1
Anneling53°C
DIAP2
Anneling50.5°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
Lanes 4 and 5;DIAP2 cDNA
Again the DNA fragments with the expected sizes were not detected.


24th, August

Member

Matsunami


We have decreased the amount of the template API2-MALT1 cDNA by diluting 10 x and 100 x , then used for the PCR reactions under the following conditions.
PCR reaction
10 µM Primer F0.4 µl
10 µM Primer R0.4 µl
Template DNA1 µl
10 x Ex Taq Buffer2 µl
Takara Ex Taq0.1 µl
2.0 mM dNTPs2 µl
ddH2O14.1 µl
 total 20 µl
Cycle
Pre-Denature94°C2min 
Denature94°C30sec37 Cycle
Anneling51.5°C30sec
Extension72°C3min20sec
+Extension72°C10min 
End4°Ckeep 
PCR products were applied to the agarose gel electrophoresis.


Results

Image of the agarose gel.


Lane 2;DNA size marker(1 Kbp ladder)
Lanes 3~6;API2-MALT1 cDNA (10 X diluted)
Lanes 8~11;API2-MALT1 cDNA(100 X diluted)
No amplified DNA was detected.


25th, August

Member

Matsunami


Again different annealing conditions were tested.
API2-MALT1
Anneling51.5°C
DIAP2
Anneling50.5°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
Lanes 4 and 5;DIAP2 cDNA
No PCR product was detected for API2-MALT1. The PCR products with the expected size (1.5 kb) were detected for DIAP2.


29th, August

Member

Takeda、Yokoigawa


The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.
DIAP2
DIAP2
PCR reaction in ddH2O44 µl
10 x H Buffer5 µl
Xho1 µl
 total 50 µl
pUAST-flag vector
pUAST-flag vector(500 ng/µl)10 µl
ddH2O34 µl
10 x H Buffer5 µl
Xho1 µl
 total 50 µl


30th, August

Member

Takeda, Yokoigawa


The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XbaI for 20 hours at 37°C.
DIAP2
PCR reaction in ddH2O44 µl
10 x M Buffer5 µl
Xba1 µl
 total 50 µl
pUAST-flag vector
pUAST-flag vector(500 ng/µl)10 μl
ddH2O34 µl
10 x H Buffer5 µl
Xba1 µl
 total 50 µl


31th, August

Member

Takeda, Yokoigawa


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

Image of agarose gel after DNA fragment isolation.


The amount of the purified pUAST-flag vector was 45.0375 ng/µl.
PCR products for DIAP2 was not successfully recovered from the agarose gel.