Difference between revisions of "Part:BBa K332011:Experience"
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''Bacillus thuringiensis subsp. Israelensis'' (BCRC15860) | ''Bacillus thuringiensis subsp. Israelensis'' (BCRC15860) | ||
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1. Prepare agar plate for Bti. | 1. Prepare agar plate for Bti. | ||
| − | Beef extract 3.0g | + | Beef extract 3.0g |
| − | + | Peptone 5.0g | |
| − | Peptone | + | Agar 15.0g |
| − | + | ddH2O 1 L | |
| − | Agar | + | |
| − | + | ||
| − | ddH2O | + | |
*adjust PH to 7.0 | *adjust PH to 7.0 | ||
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1. Extract genomic DNA of Bti. by liquid nitrogen. | 1. Extract genomic DNA of Bti. by liquid nitrogen. | ||
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2. Add some ddH2O to dilute the DNA. | 2. Add some ddH2O to dilute the DNA. | ||
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3. Design primers | 3. Design primers | ||
Vf2: ATTCAATAAAAGGTGGAATGAATTATGGA Tm: 53°C | Vf2: ATTCAATAAAAGGTGGAATGAATTATGGA Tm: 53°C | ||
| − | VR: GTGCTAACATGACTTCTACTTTAGT | + | VR: GTGCTAACATGACTTCTACTTTAGT Tm: 52.8°C |
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4. Find the best PCR condition by gradient PCR. | 4. Find the best PCR condition by gradient PCR. | ||
Anneling temperature: 51°C±10°C | Anneling temperature: 51°C±10°C | ||
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5. PCR by B-taq plus on the best condition | 5. PCR by B-taq plus on the best condition | ||
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B-taq buffer 5.0 | B-taq buffer 5.0 | ||
dNTP（2mM） 5.0 | dNTP（2mM） 5.0 | ||
| − | forward primer（10μM） | + | forward primer（10μM） 1.5 |
| − | reverse primer（10μM） | + | reverse primer（10μM） 1.5 |
| − | B-taq plus DNA polymerase（2Kb） | + | B-taq plus DNA polymerase（2Kb） 1.0 |
| − | ddH2O | + | ddH2O 34.0 |
| − | Total | + | Total 50 μl |
6. Digestion to confirm the cry11Aa fragment and enzyme sites. | 6. Digestion to confirm the cry11Aa fragment and enzyme sites. | ||
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7. TA clone | 7. TA clone | ||
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8. DNA sequencing | 8. DNA sequencing | ||
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1. Design primers by primerX. | 1. Design primers by primerX. | ||
EcoR1-Vf2: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A | EcoR1-Vf2: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A | ||
| − | EcoR1-VR: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G | + | EcoR1-VR: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G |
| − | Spe1-Vf2: ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G | + | Spe1-Vf2: ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G |
| − | Spe1-VR: CTT CCC CCA TTA ATA ACT TAT AAA CTA GTC CTC CCC ATT CAT | + | Spe1-VR: CTT CCC CCA TTA ATA ACT TAT AAA CTA GTC CTC CCC ATT CAT |
2. Digestion to confirm the fragments | 2. Digestion to confirm the fragments | ||
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1. Design primers by Assembly standard 10. | 1. Design primers by Assembly standard 10. | ||
Vf2: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACT | Vf2: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACT | ||
| − | VR: GTTTCTTC CTGCAG CGGCCGC T ACTAGT | + | VR: GTTTCTTC CTGCAG CGGCCGC T ACTAGT ACTACTTTAGTAACGGATT |
2. PCR condition | 2. PCR condition | ||
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3. Ligation to backbones(Psb1C3). | 3. Ligation to backbones(Psb1C3). | ||
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1. Thaw competent cells and BBa_K332012 plasmid on ice. | 1. Thaw competent cells and BBa_K332012 plasmid on ice. | ||
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2. Add 2ul plasmid to competent cell and place in ice for 5 minutes. | 2. Add 2ul plasmid to competent cell and place in ice for 5 minutes. | ||
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3. Put the transformed cells into 42℃ water bath for 45 seconds. | 3. Put the transformed cells into 42℃ water bath for 45 seconds. | ||
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4. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 µg/ml kanamycin. | 4. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 µg/ml kanamycin. | ||
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5. Incubate the cultures at 37°C overnight. | 5. Incubate the cultures at 37°C overnight. | ||
Revision as of 12:10, 24 October 2010
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Applications of BBa_K332011
(I) Culture Bti.
Bacillus thuringiensis subsp. Israelensis (BCRC15860)
1. Prepare agar plate for Bti.
Beef extract 3.0g Peptone 5.0g Agar 15.0g ddH2O 1 L
- adjust PH to 7.0
- No antibiotic
- Be aware of contamination
2. Plate Bti. on the medium and incubate the cultures at 30°C overnight.
(II) Clone cry11Aa from Bti. into TA vector
1. Extract genomic DNA of Bti. by liquid nitrogen.
2. Add some ddH2O to dilute the DNA.
3. Design primers
Vf2: ATTCAATAAAAGGTGGAATGAATTATGGA Tm: 53°C VR: GTGCTAACATGACTTCTACTTTAGT Tm: 52.8°C
4. Find the best PCR condition by gradient PCR.
Anneling temperature: 51°C±10°C
5. PCR by B-taq plus on the best condition
Template DNA（10ng/μl） 2.0 B-taq buffer 5.0 dNTP（2mM） 5.0 forward primer（10μM） 1.5 reverse primer（10μM） 1.5 B-taq plus DNA polymerase（2Kb） 1.0 ddH2O 34.0 Total 50 μl
6. Digestion to confirm the cry11Aa fragment and enzyme sites.
7. TA clone
8. DNA sequencing
(III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1
1. Design primers by primerX.
EcoR1-Vf2: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A EcoR1-VR: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G Spe1-Vf2: ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G Spe1-VR: CTT CCC CCA TTA ATA ACT TAT AAA CTA GTC CTC CCC ATT CAT
2. Digestion to confirm the fragments
(IV) PCR construction of Biobrick parts
1. Design primers by Assembly standard 10.
Vf2: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACT VR: GTTTCTTC CTGCAG CGGCCGC T ACTAGT ACTACTTTAGTAACGGATT
2. PCR condition
3. Ligation to backbones(Psb1C3).
(V) Transform into E.coli
1. Thaw competent cells and BBa_K332012 plasmid on ice.
2. Add 2ul plasmid to competent cell and place in ice for 5 minutes.
3. Put the transformed cells into 42℃ water bath for 45 seconds.
4. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 µg/ml kanamycin.
5. Incubate the cultures at 37°C overnight.
(VI) Culture with mosquito larvae and observe
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