Team:Shenzhen/Result/YAO.Channel
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<ul><li>III. Third Vector Construction</li></ul> | <ul><li>III. Third Vector Construction</li></ul> | ||
- | 1. Mix the strain which containing following carrier and 4 ml of LB+AMP liquid medium in 15 ml | + | <ul><p>1. Mix the strain which containing following carrier and 4 ml of LB+AMP liquid medium in 15 ml centrifuge tube, shake at 37 ℃, 220 rpm overnight.</p><p> |
- | centrifuge tube, shake at 37 ℃, 220 rpm overnight. | + | GAL-ZIM17-E0030, GAL-ZIM17-E1010, GAL-FER-E0030, GAL-FER-E1010, GAL-TIM2-E0030, GAL-TIM2-E1010, GAL-TOM22-E0030, GAL-TOM22-E1010, GAL-TOM70-E0030, GAL-TOM70-E1010, GAL-TOM20-E0030, GAL-TOM20-E1010, GAL-Tom40-E0030, GAL-Tom40-E1010(kan resistant)</p><p> |
- | GAL-ZIM17-E0030, GAL-ZIM17-E1010, GAL-FER-E0030, | + | BBa-J63002(Amp resistant, yeast ADH terminator)</p></ul> |
- | GAL-FER-E1010, GAL-TIM2-E0030, GAL-TIM2-E1010, GAL-TOM22-E0030, | + | <ul><p>2. Extract plasmid</p></ul> |
- | GAL-TOM22-E1010, GAL-TOM70-E0030, GAL-TOM70-E1010, GAL-TOM20-E0030, | + | <ul><p>3. Measure plasmid concentration</p></ul> |
- | GAL-TOM20-E1010, GAL-Tom40-E0030, GAL-Tom40-E1010(kan resistant) | + | <ul><p>4. Digestion SpeI and PstI with 10X H Buffer:</p><p> |
- | BBa-J63002(Amp resistant, yeast ADH terminator) | + | GAL-ZIM17-E0030, GAL-ZIM17-E1010, GAL-FER-E0030, GAL-FER-E1010, GAL-TIM2-E0030, GAL-TIM2-E1010, GAL-TOM22-E0030, GAL-TOM22-E1010, GAL-TOM70-E0030, GAL-TOM70-E1010, GAL-TOM20-E0030, GAL-TOM20-E1010, GAL-Tom40-E0030, GAL-Tom40-E1010</p><p> |
- | 2. Extract plasmid | + | XbaI and PstI with 10X H Buffer: </p><p> |
- | 3. Measure plasmid concentration | + | BBa-J63002</p></ul> |
- | 4. Digestion SpeI and PstI with 10X H Buffer: | + | <ul><p>5. Gel electrophoresis, recycle and measure plasmid concentration</p></ul> |
- | GAL-ZIM17-E0030, GAL-ZIM17-E1010, GAL-FER-E0030, | + | <ul><p>6. Ligation </p><p> |
- | GAL-FER-E1010, GAL-TIM2-E0030, GAL-TIM2-E1010, GAL-TOM22-E0030, | + | GAL-ZIM17-E0030, GAL-ZIM17-E1010, GAL-FER-E0030, GAL-FER-E1010, GAL-TIM2-E0030, GAL-TIM2-E1010, GAL-TOM22-E0030, GAL-TOM22-E1010, GAL-TOM70-E0030, GAL-TOM70-E1010, GAL-TOM20-E0030, GAL-TOM20-E1010, GAL-Tom40-E0030, GAL-Tom40-E1010 2ul respectively ligating with BBa-J63002 6ul.</p></ul> |
- | GAL-TOM22-E1010, GAL-TOM70-E0030, GAL-TOM70-E1010, GAL-TOM20-E0030, | + | <ul><p>7. Transformation</p></ul> |
- | GAL-TOM20-E1010, GAL-Tom40-E0030, GAL-Tom40-E1010 | + | <ul><p>8. PCR</p></ul> |
- | XbaI and PstI with 10X H Buffer: | + | <ul><p>9. Shake culture</p></ul> |
- | BBa-J63002 | + | <ul><p>10. Sequencing</p></ul> |
- | 5. Gel electrophoresis, recycle and measure plasmid concentration | + | <ul><p>11. Named the correct vectors( for convenient description) </p><p> |
- | 6. Ligation | + | GAL-ZIM17-E0030-J63002, GAL-ZIM17-E1010-J63002, GAL-FER-E0030-J63002, GAL-FER-E1010-J63002, GAL-TIM2-E0030-J63002, GAL-TIM2-E1010-J63002, GAL-TOM22-E0030-J63002, GAL-TOM22-E1010-J63002, GALTOM70-E0030-J63002, GAL-TOM70-E1010-J63002, GAL-TOM20-E0030-J63002, GALTOM20-E1010-J63002, GAL-Tom40-E0030-J63002, GAL-Tom40-E1010-J63002</p></ul> |
- | GAL-ZIM17-E0030, GAL-ZIM17-E1010, GAL-FER-E0030, | + | |
- | GAL-FER-E1010, GAL-TIM2-E0030, GAL-TIM2-E1010, GAL-TOM22-E0030, | + | |
- | GAL-TOM22-E1010, GAL-TOM70-E0030, GAL-TOM70-E1010, GAL-TOM20-E0030, | + | |
- | GAL-TOM20-E1010, GAL-Tom40-E0030, GAL-Tom40-E1010 | + | |
- | + | ||
- | 7. Transformation | + | |
- | 8. PCR | + | |
- | 9. Shake culture | + | |
- | 10. Sequencing | + | |
- | 11. Named the correct vectors( for convenient description) | + | |
- | GAL-ZIM17-E0030-J63002, GAL-ZIM17-E1010-J63002, GAL- | + | |
- | + | ||
- | GAL-TIM2-E1010-J63002, GAL-TOM22-E0030-J63002, GAL-TOM22-E1010-J63002, GALTOM70-E0030-J63002, GAL-TOM70-E1010-J63002, GAL-TOM20-E0030-J63002, GALTOM20-E1010-J63002, GAL-Tom40-E0030-J63002, GAL-Tom40-E1010-J63002 | + | |
<ul><li>IV. Construction of yeast expression vector</li></ul> | <ul><li>IV. Construction of yeast expression vector</li></ul> | ||
- | 1. Mix the strain which containing following carrier and 4 ml of LB+AMP liquid medium in 15 ml | + | <ul><p>1. Mix the strain which containing following carrier and 4 ml of LB+AMP liquid medium in 15 ml centrifuge tube, shake at 37 ℃, 220 rpm overnight</p><p> |
- | centrifuge tube, shake at 37 ℃, 220 rpm overnight | + | GAL-ZIM17-E0030-J63002, GAL-ZIM17-E1010-J63002, GAL-FER-E0030-J63002, GAL-FER-E1010-J63002, GAL-TIM2-E0030-J63002, GAL-TIM2-E1010-J63002, GAL-TOM22-E0030-J63002, GAL-TOM22-E1010-J63002, GALTOM70-E0030-J63002, GAL-TOM70-E1010-J63002, GAL-TOM20-E0030-J63002, GALTOM20-E1010-J63002, GAL-Tom40-E0030-J63002, GAL-Tom40-E1010-J63002(kan resistant)</p><p> |
- | GAL-ZIM17-E0030-J63002, GAL-ZIM17-E1010-J63002, GAL- | + | YEP352(Amp resistant, yeast ADH terminator)</p></ul> |
- | + | <ul><p>2. Extract plasmid</p></ul> | |
- | GAL-TIM2-E1010-J63002, GAL-TOM22-E0030-J63002, GAL-TOM22-E1010-J63002, GALTOM70-E0030-J63002, GAL-TOM70-E1010-J63002, GAL-TOM20-E0030-J63002, GALTOM20-E1010-J63002, GAL-Tom40-E0030-J63002, GAL-Tom40-E1010-J63002(kan | + | <ul><p>3. Measure plasmid concentration</p></ul> |
- | resistant) | + | <ul><p>4. Digestion </p><p> |
- | YEP352(Amp resistant, yeast ADH terminator) | + | XbaI and PstI:</p><p> |
- | 2. Extract plasmid | + | GAL-ZIM17-E0030-J63002, GAL-ZIM17-E1010-J63002, GAL-FER-E0030-J63002, GAL-FER-E1010-J63002, GAL-TIM2-E0030-J63002, GAL-TIM2-E1010-J63002, GAL-TOM22-E0030-J63002, GAL-TOM22-E1010-J63002, GALTOM70-E0030-J63002, GAL-TOM70-E1010-J63002, GAL-TOM20-E0030-J63002, GAL-TOM20-E1010-J63002, GAL-Tom40-E0030-J63002, GAL-Tom40-E1010-J63002, YEP352</p></ul> |
- | 3. Measure plasmid concentration | + | <ul><p>5. Gel electrophoresis, recycle and measure plasmid concentration</p></ul> |
- | 4. Digestion | + | <ul><p>6. Ligation </p><p> |
- | XbaI and PstI: | + | GAL-ZIM17-E0030-J63002, GAL-ZIM17-E1010-J63002, GAL-FER-E0030-J63002, GAL-FER-E1010-J63002, GAL-TIM2-E0030-J63002, GAL-TIM2-E1010-J63002, GAL-TOM22-E0030-J63002, GAL-TOM22-E1010-J63002, GAL-TOM70-E0030-J63002, GAL-TOM70-E1010-J63002, GAL-TOM20-E0030-J63002, GAL-TOM20-E1010-J63002, GAL-Tom40-E0030-J63002, GAL-Tom40-E1010-J63002 6ul respectively ligating with YEP532 2ul.</p></ul> |
- | GAL-ZIM17-E0030-J63002, GAL-ZIM17-E1010-J63002, GAL- | + | <ul><p>7. Transformation</p></ul> |
- | + | <ul><p>8. PCR</p></ul> | |
- | GAL-TIM2-E1010-J63002, GAL-TOM22-E0030-J63002, GAL-TOM22-E1010-J63002, GALTOM70-E0030-J63002, GAL-TOM70-E1010-J63002, GAL-TOM20-E0030-J63002, GAL-TOM20-E1010-J63002, GAL-Tom40-E0030-J63002, GAL-Tom40-E1010-J63002, YEP352 | + | <ul><p>9. Shake culture</p></ul> |
- | 5. Gel electrophoresis, recycle and measure plasmid concentration | + | <ul><p>10. Sequencing</p></ul> |
- | 6. Ligation | + | <ul><p>11. Named the correct vectors( for convenient description)</p><p> |
- | GAL-ZIM17-E0030-J63002, GAL-ZIM17-E1010-J63002, GAL- | + | YEP-GAL-ZIM17-E0030-J63002, YEP-GAL-ZIM17-E1010-J63002, YEPGAL-FER-E0030-J63002, YEP-GAL-FER-E1010-J63002, YEP-GALTIM2-E0030-J63002, YEP-GAL-TIM2-E1010-J63002, YEP-GAL-TOM22-E0030-J63002, YEP-GAL-TOM22-E1010-J63002, YEP-GAL-TOM70-E0030-J63002, YEP-GAL-TOM70-E1010-J63002, YEP-GAL-TOM20-E0030-J63002, YEP-GAL-TOM20-E1010-J63002, YEPGAL-Tom40-E0030-J63002, YEP-GAL-Tom40-E1010-J63002</p></ul> |
- | + | ||
- | GAL-TIM2-E1010-J63002, GAL-TOM22-E0030-J63002, GAL-TOM22-E1010-J63002, | + | |
- | GAL-TOM70-E0030-J63002, GAL-TOM70-E1010-J63002, GAL-TOM20-E0030-J63002, | + | |
- | GAL-TOM20-E1010-J63002, GAL-Tom40-E0030-J63002, GAL-Tom40-E1010-J63002 6ul | + | |
- | respectively ligating with YEP532 2ul. | + | |
- | 7. Transformation | + | |
- | 8. PCR | + | |
- | 9. Shake culture | + | |
- | 10. Sequencing | + | |
- | 11. Named the correct vectors( for convenient description) | + | |
- | YEP-GAL-ZIM17-E0030-J63002, YEP-GAL-ZIM17-E1010-J63002, YEPGAL-FER-E0030-J63002, YEP-GAL-FER-E1010-J63002, YEP-GALTIM2-E0030-J63002, YEP-GAL-TIM2-E1010-J63002, YEP-GAL-TOM22-E0030-J63002, | + | |
- | YEP-GAL-TOM22-E1010-J63002, YEP-GAL-TOM70-E0030-J63002, YEP-GAL-TOM70- | + | |
- | E1010-J63002, YEP-GAL-TOM20-E0030-J63002, YEP-GAL-TOM20-E1010-J63002, YEPGAL-Tom40-E0030-J63002, YEP-GAL-Tom40-E1010-J63002 | + | |
<ul><li>V. Yeast transformation</li></ul> | <ul><li>V. Yeast transformation</li></ul> | ||
- | 1. Extract plasmid | + | <ul><p>1. Extract plasmid</p><p> |
- | YEP-GAL-ZIM17-E0030-J63002, YEP-GAL-ZIM17-E1010-J63002, YEPGAL-FER-E0030-J63002, YEP-GAL-FER-E1010-J63002, YEP-GALTIM2-E0030-J63002, YEP-GAL-TIM2- | + | YEP-GAL-ZIM17-E0030-J63002, YEP-GAL-ZIM17-E1010-J63002, YEPGAL-FER-E0030-J63002, YEP-GAL-FER-E1010-J63002, YEP-GALTIM2-E0030-J63002, YEP-GAL-TIM2-1010-J63002, YEP-GAL-TOM22-E0030-J63002, </p><p> |
- | YEP-GAL-TOM22-E1010-J63002, YEP-GAL-TOM70-E0030-J63002, YEP-GAL-TOM70- | + | YEP-GAL-TOM22-E1010-J63002, YEP-GAL-TOM70-E0030-J63002, YEP-GAL-TOM70-E1010-J63002, YEP-GAL-TOM20-E0030-J63002, YEP-GAL-TOM20-E1010-J63002, YEPGAL-Tom40-E0030-J63002, YEP-GAL-Tom40-E1010-J63002</p></ul> |
- | E1010-J63002, YEP-GAL-TOM20-E0030-J63002, YEP-GAL-TOM20-E1010-J63002, YEPGAL-Tom40-E0030-J63002, YEP-GAL-Tom40-E1010-J63002 | + | <ul><p>2. Preparation of yeast competent</p><p> |
- | 2. Preparation of yeast competent | + | (1) Streak a YPDA agar plate with a small portion of AH109 or Y187. Incubate the plate upside down at 30°C until colonies appear(~3days). Yeast strains can be stored for up to 1 month at 4°C on YPDA medium in culture plates.</p><p> |
- | (1) | + | (2) Prepare 1.1X TE/LiAc Solution.</p><p> |
- | Y187. Incubate the plate upside down at 30°C until colonies appear(~3days). Yeast | + | (3) Prepare YPDA liquid medium (Yeast Protocols Handbook).</p><p> |
- | strains can be stored for up to 1 month at 4°C on YPDA medium in culture | + | (4) Inoculate one colony(<4 weeks old, 2–3mm in diameter) into 3ml of YPDA medium in a sterile, 15-ml centrifuge tube.</p><p> |
- | plates | + | (5) Incubate at 30°C with shaking for 8hr.</p><p> |
- | (2) | + | (6) Transfer 5µl of the culture to a 250-ml flask containing 50ml of YPDA.</p><p> |
- | (3) | + | (7) Incubate at 30°C with shaking at 230–250 rpm for 16–20hr.The OD600 should reach 0.15–0.3.</p><p> |
- | (4) | + | (8) Centrifuge the cells at 700 x g for 5min at room temperature.</p><p> |
- | medium in a sterile, 15-ml centrifuge tube. | + | (9) Discard the supernatant and resuspend the cell pellet in 100 ml of YPDA.</p><p> |
- | (5) | + | (10) Incubate at 30°C for 3–5hr(OD600= 0.4–0.5).</p><p> |
- | (6) | + | (11) Centrifuge the cells at 700 x g for 5min at room temperature.</p><p> |
- | (7) | + | (12) Discard the supernatant and resuspend the cell pellet in 60ml of sterile, deionized H2O.</p><p> |
- | 0.15–0.3. | + | (13) Centrifuge the cells at 700 x g for 5min at room temperature.</p><p> |
- | (8) | + | (14) Discard the supernatant and resuspend the cells in 3 ml of 1.1 X TE/LiAc Solution.</p><p> |
- | (9) | + | (15) Split the resuspension between two 1.5-ml microcentrifuge tubes (1.5 ml pertube).</p><p> |
- | (11) | + | (16) Centrifuge each tube at high speed for 15 sec.</p><p> |
- | (12) | + | (17) Discard the supernatant and resuspend each pellet in 600 µl of 1.1 X TE/LiAc Solution.</p></ul> |
- | deionized H2O. | + | <ul><p>3. Transformation</p><p> |
- | (13) | + | (1) 1.5ml centrifuge:</p><p> |
- | (14) | + | plasmid 0.2ug</p><p> |
- | (15) | + | Sperm DNA*(10 mg/ml) 0.1mg</p><p> |
- | + | * Sperm DNA When freshly prepared water bath boil for 20 minutes, and immediately inserted into the ice bath and stored at -20 °C</p><p> | |
- | (16) | + | (2) Add 100 ul with 1 × TE / LiAc resuspended yeast competent cells per tube, mixed with shaking;</p><p> |
- | (17) | + | (3) Each added 600ul PEG / LiAc, shaken vigorously (to improve the conversion efficiency), 30 °C, 200 rpm shaking culture 30 min; </p><p> |
- | Solution. | + | (4) Each adding 70 ul DMSO, slowly inverted mix (not oscillating), 42 °C water bath thermal shock 15 min, insert into the ice bath to cool 1-2 min; </p><p> |
- | 3. Transformation | + | (5) 14,000rpm × 5 sec, discard supernatant, resuspend cells with 0.1 ml 1×TE, overlay on SD/-Ura solid medium, 30°C inverted culture 3 days in incubator.</p></ul> |
- | (1) | + | <ul><p>4. Check out</p><p> |
- | plasmid | + | Draw the colonies grown on the said plate in the galactose-containing SD/-Ura solid plates, until the colonies grow, single colonies were picked, and microscopic examination under a fluorescence microscope and record the results.</p></ul></div> |
- | Sperm DNA*(10 mg/ml) | + | |
- | *Sperm DNA When freshly prepared water bath boil for 20 minutes, and immediately inserted | + | |
- | into the ice bath and stored at -20 °C | + | |
- | (2) | + | |
- | shaking; | + | |
- | (3) | + | |
- | 30 °C, 200 rpm shaking culture 30 min; | + | |
- | (4) | + | |
- | shock 15 min, insert into the ice bath to cool 1-2 min; | + | |
- | (5) | + | |
- | Ura solid medium, 30°C inverted culture 3 days in incubator. | + | |
- | 4. Check out | + | |
- | Draw the colonies grown on the said plate in the galactose-containing SD/-Ura solid plates, | + | |
- | until the colonies grow, single colonies were picked, and microscopic examination under a | + | |
- | fluorescence microscope and record the results. | + | |
</div> | </div> | ||
{{:Team:Shenzhen/Temp/gallery.htm}} | {{:Team:Shenzhen/Temp/gallery.htm}} |
Revision as of 08:36, 25 September 2012