Team:KIT-Kyoto/Notebook-week2

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<h2>August 8th</h2>
<h2>August 8th</h2>

Latest revision as of 04:13, 26 September 2012

August 8th



We isolated 5 colonies from the LB plate, and cultured.


August 9th



1. Purification of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.
We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.


2. Characterization of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.

　Each diluted DNA sample　　1μL　
　10×H buffer　　0.5μL　
　EcoRⅠ　　0.2μL　
　dH2O　　3.3μL　
　total　　5μL　


After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.
Left,　10fold dilution,　2uL marker,　20fold dilution,　30fold dilution,　1uL marker,　40fold dilution,　0.5uL marker right.


Result


The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.


3. PCR amplification of the insert DNA

The four 5'primers were designed for PCR as follows.

・GW3r
5’-ATCGAGGCCTGTCTAGAGAAGC
・TNFA-1
5’-GGCTTTGCTATGATACTCGGAACTG
・TNFA-2
5’-GTAAAATGTGAAACGCCCAACTGC
・TNFA-3
5’-GGACTCCAGAAAACAAGGGCTTT


The 3’ primer was designed as follows.
・SVr
5’-GGCATTCCACCACTGCTCCC


PCR reactions with the following combinations of primers were carried out.
sample１→GW3r―SVr
sample２→TNFA-1―SVr
sample３→TNFA-2―SVr
sample４→TNFA-3―SVr


PCR was carried out in the following reactions.
　　　Each sample　
　50ng/μL LR TNFA-1　　1μL　
　10×rTaq buffer　　2μL　
　2mM dNTPs　　2μL　
　25mM MgCl2　　0.8μL　
　10P 5'primer　　0.6μL　
　10P 3'primer　　0.6μL　
　rTaq　　0.4μL　
　dH2O　　12.6μL　
　total　　20μL　


Reaction conditions of PCR
　temperature　　time　　cycle　
　95°C　　2min　　　
　95°C　　15sec　　25cycle　
　55°C　　30sec　　25cycle　
　68°C　　2min30sec　　25cycle　
　68°C　　2min30sec　　　
　14°C　　∞　　　

August 10th



Electrophoresis of PCR products was carried out.
After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.

Left　marker5uL　sample１　sample２　sample３　sample４　Wright

Result

The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.


August 13th



Cut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction.

The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.

　the candidate pENTR-TNFAIP3 prepared on 8/6　　1μL　
　10×M buffer　　0.5μL　
　Hind Ⅲ　　0.2μL　
　dH2O　　3.3μL　
　total　　5μL　


After reaction, samples were electrophoresed in following order.

Left: 1kb marker5uL cut sample
Right: uncut sample


Results


Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful.


August 14th



Based on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again.

Composition of the reaction
　　　sample1　
　10ng/μL TNFAIP3　　6μL　
　10×KOD plus buffer　　10μL　
　2mM dNTPs　　10μL　
　25mM MgSO4　　3.2μL　
　10P 5'primer　　3μL　
　10P 3'primer　　3μL　
　KOD plus　　2μL　
　dH2O　　62.8μL　
　total　　100μL　


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


PCR product was applied to the 0.7% agarose gel electrophoresis.

Results



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