Team:KIT-Kyoto/Notebook-week3
From 2012.igem.org
August 20th1. Isolation of DNA fragment from the gel Sample applied to the electrophoresis Composition
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 21st1. 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
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 22ndWe isolated 6 colonies from a plate and incubated in 2.5mL of Kanamycin(+) LB liquid culture medium for 16 hours. August 23rd1 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
Reaction
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.
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 24thSingle 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 25th1. 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
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 26thThe female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately. |