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Team:Todai-Tokyo/Notebook/LDLR
From 2009.igem.org
Contents |
Plan
Aim: Create yeast cells that express LDLR on their cell membrane
Methods:
- Clone the LDLR gene.
- Create biobrick of LDLR.
7/6
Overview:
1.PCR LDLR gene from plasmid containing LDLR cDNA
2.gel-purify DNA from the PCR product
3.insert the DNA to vector (iGEM parts)
4.transform into E. coli and select for ampicillin resistance
5.check for transformation success using colony PCR by LDLR primers
Constructs to be created:
pGal1-Kozak-LDLR-terminator
Obtaining DNA:
Resuspended DNA in the following wells with 10ul water:
plate 1 7D
pGal1(including Kozak sequence)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_J63006 Gal1 promoter]
Transformed 1ul of each of the above into DH5a competent cells:
Transformation
- Mix 1ul of DNA with 100ul of competent cells on ice.
- Leave on ice for 30 minutes.
- Heat shock at 42℃ for 45 seconds.
- Leave on ice for 2 minutes.
- Add 500ul of LB and incubate at 37℃ for 1 hour.
- Plate on LB-ampicillin plates.
7/7
Miniprep of E.coli cells containing LDLR gene with Promega, Wizard Plus SV Miniprep DNA Purification System
Successful
7/19
PCR of LDLR
0.4ul 50uM 5’primer
0.4ul 50uM 3’primer
1.6ul 2.5mM dNTP
2ul 10×Pfu Ultra 2 buffer
0.05ul LDLR plasmid
0.5ul Pfu Ultra
15.05ul MilliQ water
↓
Performed PCR using the following program:
1. 95℃ 2min
2. 95℃ 30sec
3. 55℃ 30sec
4. 72.5℃ 60sec
5. repeat 2-4 29times
6. 25℃ ∞
PCR successful
We cut the region of LDLR out of the gel and purified PCR product using Promega kit.
infusion of LDLR
4ul PCR product (12.35ug/ml)
3ul vector (iGEM parts plate1 D-3, 14.95ug/ml)
2ul 5×infusion reaction buffer
1ul infusion enzyme
↓
37℃ 15min
50℃ 15min
↓
TE buffer up to 20ul
↓
added 10ul of the sample to DH5α (090614) and put on ice for 15 min.
↓
42℃ 45sec
↓
added 500ul LB broth to the tube.
↓
placed it on LB ampicilin plate.
7/20
colony PCR
put a small amount of single colony into each tube with 5ul MilliQ water
↓
95℃ 5min
↓
PCR reaction
1ul 10×buffer
0.8ul 2.5mMdNTP
0.08ul Ex-Taq
0.1ul 5’-primer
0.1ul 3’-primer
2.92ul MilliQ water
↓
added the PCR reaction to each tube.
↓
Performed PCR using the following program:
1. 95℃ 2min
2. 95℃ 30sec
3. 55℃ 30sec
4. 72.5℃ 120sec
5. repeat 2-4 29times
6. 25℃ ∞
PCR unsuccessful・・・.
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