Team:KIT-Kyoto/Notebook-week3

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<h2>August 20th</h2>
<h2>August 20th</h2>

Revision as of 04:13, 26 September 2012

August 20th


1. Isolation of DNA fragment from the gel Sample applied to the electrophoresis

Composition
　a product of PCR attB TNFAIP3(8/1)　
　DNA sample　　　　　　　　90μL
　6×Dye　　　　　　　　18μL
　Total　　　　　　　　108μL


Results



DNA was isolated from the agarose gel by QIA Quick Gel Extraction Kit, then the DNA was dissolved in 40 uL of TE.

August 21st


1. Measuring the concentration of the attB TNFAIP3 DNA fragment
The order of sample applied to the electrophoresis
1kbmarker3uL，1uL of 5 fold dilution of attB TNFAIP3, 1uL of 10 fold dilution of attB TNFAIP3 DNA fragment，2uL of 1kbmarker, 1uL of 15 fold dilution of attB TNFAIP3 DNA fragment, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragment, 1kbmarker1uL,

Result



The concentration of the isolated attB TNFAIP3 DNA fragments was estimated to be 35ng/uL.



2. BP reaction
　1.5mL tube
　attB TNFAIP3(35ng/μL)　2μL　
　pDONR(150ng/μL)　　1μL　
　TE buffer　　5μL　
　Total　　8μL　


We added 2uL of BP Clonase Ⅱ enzyme mix to this solution and incubate it for 2 hour.

3. Transformation
　We added 100uL of XL1-Blue to the BP reaction products to do transformation.

August 22nd


We isolated 6 colonies from a plate and incubated in 2.5mL of Kanamycin(＋) LB liquid culture medium for 16 hours.

August 23rd


1 Purification of the candidate pENTR-TNFAIP3 DNA
We purified the candidate pENTR-TNFAIP3 DNA fro six independent colonies cultured on August 22 using QIA prep Spin Miniprep

2 Characterization of the candidate pENTR-TNFAIP3 DNA
We conducted PCR on the following condition to confirm the pENTR-TNFAIP3 DNA using primer designed for attB

Composition
　BP TNFAIP3　　0.2μL　
　10× rTaq buffer　　0.8μL　
　2mM dNTPs　　2μL　
　25mM MgCl2　　2μL　
　10P 5'primer　　0.6μL　
　10P 3'primer　　0.6μL　
　rTaq　　0.4μL　
　dH2O　　13.4μL　
　Total　　20μL　


Reaction
　BP TNFAIP3　　0.2μL　
　10× rTaq buffer　　0.8μL　
　2mM dNTPs　　2μL　
　25mM MgCl2　　2μL　
　10P 5'primer　　0.6μL　
　10P 3'primer　　0.6μL　
　rTaq　　0.4μL　
　dH2O　　13.4μL　
　Total　　20μL　


　temperature　　time　　cycle　
　95°C　　2min.　　　
　95°C　　15sec　　25cycle　
　60°C　　30sec　　25cycle　
　68°C　　2min30sec　　25cycle　
　68°C　　2min30sec　　　
　14°C　　∞　　　


The PCRproducts were applied to agarose gel electrophoresis
From left to right: pDONR DNA 1uL, ６the candidate pENTR-TNFAIP3 2uL each

Photo of agarose gel


The amplified DNA fragments were detected in the gel.

3. LR reaction
LR reactions were carried out under conditions as described below.

　pENTR-TNFAIP3 (prepared on 8/23)　　1μL　
　pTFW or pTGW (Destination vectors)　　0.5μL　
　TE Buffer　　6.5μL　
　Total　　8μL　


2uL LR clonaseⅡ enzyme mix was added to the reaction and incubated for 2.5 hour.

4. The LR reaction products were transformed into E. coli XL1-Blue100uL according to the protocol and spread on the LB ampicillin(+) plate and cultured for 16 hours at 37℃.

August 24th


Single colonies were isolated from a plate with the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 and cultured them in LB ampicillin(+) liquid medium for 16 hours at 37℃.

August 25th


1. Purification of the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 DNA
the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 were purified by QIA prep Spin Miniprep Kit.

2. Characterization of the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3
We used the primer attB to confirm the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 for PCR reactions under conditions described below.

Composition
　pTFW- or pTGW-TNFAIP3　　0.2μL　
　10× rTaq buffer　　0.8μL　
　2mM dNTPs　　2μL　
　25mM MgCl2　　2μL　
　10P 5'primer　　0.6μL　
　10P 3'primer　　0.6μL　
　rTaq　　0.4μL　
　dH2O　　13.4μL　
　Total　　20μL　


　temperature　　time　　cycle　
　95°C　　2min.　　　
　95°C　　15sec　　25cycle　
　60°C　　30sec　　25cycle　
　68°C　　2min30sec　　25cycle　
　68°C　　2min30sec　　　
　14°C　　∞　　　


Photo of agarose gel


Results: Since the amplified DNAs with appropriate sizes were detected on the gel, pTFW-TNFAIP3 and pTGW-TNFAIP3 were appeared to be successfully constructed by the Gateway system ! At this point we renamed the pTFW-TNFAIP3 as pUAS-flag-TNFAIP3.

August 26th


The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately.

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