Difference between revisions of "Part:BBa K332011:Experience"
Chialemeng (Talk | contribs) |
Chialemeng (Talk | contribs) (→Applications of BBa_K332011) |
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Agar 15.0g | Agar 15.0g | ||
ddH2O 1 L | ddH2O 1 L | ||
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*adjust pH to 7.0 | *adjust pH to 7.0 | ||
*No antibiotic | *No antibiotic | ||
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6. TA cloning | 6. TA cloning | ||
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7. Digestion to confirm the cry11Aa fragment and enzyme sites. | 7. Digestion to confirm the cry11Aa fragment and enzyme sites. | ||
[[Image:cry digestion.jpg]] | [[Image:cry digestion.jpg]] | ||
lane 1: 1Kb marker | lane 1: 1Kb marker | ||
lane 2: plasmid uncut | lane 2: plasmid uncut | ||
− | lane 3: Not1 digestion(~2981bp,1966bp)[There are two Not1 | + | lane 3: Not1 digestion(~2981bp,1966bp) |
+ | [There are two Not1 sites on TA vector.] | ||
lane 4: EcoR1 digestion(~2997bp,1695bp,237bp) | lane 4: EcoR1 digestion(~2997bp,1695bp,237bp) | ||
− | [There are two EcoR1 | + | [There are two EcoR1 sites on TA vector and another EcoR1 site on cry11Aa.) |
8. DNA sequencing | 8. DNA sequencing | ||
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(VI) Culture with mosquito larvae and observe | (VI) Culture with mosquito larvae and observe | ||
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===User Reviews=== | ===User Reviews=== |
Revision as of 01:03, 30 October 2010
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
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
cry forward: ATTCAATAAAAGGTGGAATGAATTATGGA Tm: 53°C cry reverse: GTGCTAACATGACTTCTACTTTAGT Tm: 52.8°C
4. Find the best PCR condition by gradient PCR.
Anneling temperature: 51°C±10°C lane 1: 1Kb marker lane 2~13: cry11Aa PCR product(51°C±10°C) length:2Kb
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 94°C 5 min 94°C 30 sec 53°C 30 sec 72°C 2.5 min 72°C 10 min cycles:25
In the last two lanes are cry11Aa gene. The length is about 2Kb.
6. TA cloning
7. Digestion to confirm the cry11Aa fragment and enzyme sites.
lane 1: 1Kb marker lane 2: plasmid uncut lane 3: Not1 digestion(~2981bp,1966bp) [There are two Not1 sites on TA vector.] lane 4: EcoR1 digestion(~2997bp,1695bp,237bp) [There are two EcoR1 sites on TA vector and another EcoR1 site on cry11Aa.)
8. DNA sequencing
(III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1
1. Design primers by primerX.
EcoR1-f: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A EcoR1-R: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G Spe1-f: ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G Spe1-R: 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.
prefix: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACTTTAAG suffix: GTTTCTTC CTGCAG CGGCCGC T ACTAGT ACTACTTTAGTAACGGATTAATTTGC
2. PCR condition
95°C 30 sec 95°C 30 sec 55°C 1 min 72°C 2.5 min 72°C 10 min cycles:15
3. Ligation to backbones(Psb1C3).
4. Digestion to confirm the fragments
lane 1: 1Kb marker lane 2: plasmid(~3Kb) lane 3: EcoR1 digestion(~4Kb) lane 4: Spe1 digestion(~4Kb) lane 5: Pst1 digestion(~4Kb) we successfully removed EcoR1 & Spe1 enzyme sites, and added EXSP site at ends of the cry11Aa fragment.
(V) Transform into E.coli
1. Thaw competent cells and BBa_K332011 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.
6. colony PCR to confirm the fragment size (by prefix & suffix primers)
lane 1: 1kb marker lane 2: plasmid(cry11Aa+psb1C3) lane 3: PCR product(EXSP+cry11Aa) (~2Kb)
7. DNA sequencing:
(VI) Culture with mosquito larvae and observe
User Reviews
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