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 F1.5 µl
10 µM Primer R1.5 µl
Template DNA1 µl
10 x PCR Buffer5 µl
dNTPs5 µl
MgSO42 µl or 4 µl
ddH2O33 µl or 31 µl
KOD+ polymelase1 µl
 total 50 µl
Cycle
Pre-Denature94°C2min 
Denature94°C15sec35 Cyle
Anneling50.5°C30sec
Extension68°C100sec
End4°Ckeep 
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;DIAP2 cDNA
Amplified DNA fragments were barely detectable for the DIAP2.



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 ddH2O44 μl
10 x H Buffer5 μl
Xho1 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 ddH2O44 μl
10 x M Buffer5 μl
Xba1 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.


12nd, 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 F1.5 µl
10 µM Primer R1.5 µl
Template DNA1 µl
10 x PCR Buffer5 µl
dNTPs5 µl
MgSO42 µl
ddH2O33 µl
KOD+ polymelase1 µl
 total 50 µl
Cycle
Pre-Denature94°C2min 
Denature94°C15sec35 Cycle
Anneling50.5°C30sec
Extension68°C1min40sec
End4°Ckeep 


Again different annealing conditions were tested.
Annealing 50.5°C


13rd, 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  3131 bp


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 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 DIAP2 cDNA, PCR reactions were carried out by using under the following conditions.
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
Anneling53°C30sec
Extension72°C3min20sec
+Extension72°C10min 
End4°Ckeep 


DIAP2
PCR reaction
10 µM Primer F1.5 µl
10 µM Primer R1.5 µl
Template DNA1 µl
10 x PCR Buffer for KOD Plus5 µl
dNTPs5 µl
MgSO42 µl
ddH2O33 µl
KOD Plus1 µl
 total 50 µl
Cycle
Pre-Denature94°C2min 
Denature94°C15sec35 Cycle
Anneling50.5°C30sec
Extension68°C1min 40sec
End4°Ckeep 
PCR products were applied to the agarose gel electrophoresis.


The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.
DIAP2
PCR products in ddH2O44 µl
10 x H Buffer5 µl
Xho1 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
Amplified DNA fragments were barely detectable for the DIAP2.
Size of the amplified fragments for API2-MALT1 was different from the expected size


16th, September

Members

Matsunami,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 products in ddH2O44 µl
10 x M Buffer5 µl
Xba1 µl
 total 50 µl


17th, September

Members

Matsunami,Yokoigawa


The restriction enzyme-digested PCR fragments 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.


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


18th, September

Member

Matsunami


I have streaked E.coli DH5 alpha on LB plate and incubated for 16h.


19th, September

Member

Matsunami


I have picked a single colony up, transferred it into 250 ml of SOB medium in a flask.
I have incubated it at 18°C with shaking.



21st, September

Member

Matsunami


1.SOB medium in a flask was quantified by measuring the absorbance at OD600.
2. I have repeated the PCR reactions to amplify DIAP2 cDNA under the same conditions as those carried out on 12th September.
PCR reaction
10 µM Primer F1.5 µl
10 µM Primer R1.5 µl
Template DNA1 µl
10 x PCR Buffer5 µl
dNTPs5 µl
MgSO42 µl
ddH2O33 µl
KOD+ polymelase1 µl
 total 50 µl
Cycle
Pre-Denature94°C2min 
Denature94°C15sec35 Cycle
Anneling50.5°C30sec
Extension68°C1kb/min
End4°Ckeep 
Amplified DNA fragments were detectable for the DIAP2.


The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.
DIAP2
PCR products in ddH2O44 µl
10 x H Buffer5 µl
Xho1 µl
 total 50 µl


Results

1. The absorbance OD600 was over at 0.9.
2.Image of agarose gel.


Lane 1;DNA size marker(1 Kbp ladder)
Lanes 2~7;DIAP2 cDNA


22nd, September

Member

Matsunami


The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XbaI for 20 hours at 37°C.
DIAP2
PCR products in ddH2O44 µl
10 x M Buffer5 µl
Xba1 µl
 total 50 µl


23rd, September

Member

Matsunami


The restriction enzyme-digested PCR fragments 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.


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


25th, September

Member

Matsunami


I have picked a single colony up, transferred it into 250 ml of SOB medium in a flask.
I have incubated it at 18°C with shaking.



27th, September

Member

Matsunami


I have made competent cell DH5 alpha.


Results

Competency was very low.