Difference between revisions of "Team:Hong Kong-CUHK/Notebook"
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8. Restriction of BBa_K1648005, ligase into BBa_K1648004 and transform into dh5α, forming BBa_K1648006<br> | 8. Restriction of BBa_K1648005, ligase into BBa_K1648004 and transform into dh5α, forming BBa_K1648006<br> | ||
9. Sending BBa_K1648004, BBa_K1648005, BBa_K1648006 to sequencing<br> | 9. Sending BBa_K1648004, BBa_K1648005, BBa_K1648006 to sequencing<br> | ||
| − | 10. Mutated oprF is synthesized by IDT and make the PCR product of oprF from <i>Azotobacter vinelandii</i><br> | + | 10. Mutated <i>oprF</i> is synthesized by IDT and make the PCR product of <i>oprF</i> from <i>Azotobacter vinelandii</i><br> |
11. Make LB, B, BN, BN-MO plates with antibiotics -Ampicillin, kanamycin and chloramphenicol<br> | 11. Make LB, B, BN, BN-MO plates with antibiotics -Ampicillin, kanamycin and chloramphenicol<br> | ||
12. Make LB brooth and BN medium<br> | 12. Make LB brooth and BN medium<br> | ||
| Line 86: | Line 86: | ||
1. Introducing PCR product of <i>mamHIEJKLMN</i> into transformed <i>A. vinelandii</i> <br> | 1. Introducing PCR product of <i>mamHIEJKLMN</i> into transformed <i>A. vinelandii</i> <br> | ||
2. Screening of the homologous-recombinant<i> A. vinelandii</i><br> | 2. Screening of the homologous-recombinant<i> A. vinelandii</i><br> | ||
| − | 3. Restriction of PCR product of oprF and pSB1C3, ligase into C backbone and transform into dh5α, forming BBa_K1648045<br> | + | 3. Restriction of PCR product of <i>oprF</i> and pSB1C3, ligase into C backbone and transform into dh5α, forming BBa_K1648045<br> |
| − | 4. Restriction of PCR product of oprF with BBa_J13002 in the front, ligase into A backbone and transform into dh5α, forming BBa_K1648048<br> | + | 4. Restriction of PCR product of <i>oprF</i> with BBa_J13002 in the front, ligase into A backbone and transform into dh5α, forming BBa_K1648048<br> |
5. Restriction of synthesized mutated <i>oprF</i> with BBa_R0040 in the front, ligase into C backbone and transform into dh5α, forming BBa_K1648049<br> | 5. Restriction of synthesized mutated <i>oprF</i> with BBa_R0040 in the front, ligase into C backbone and transform into dh5α, forming BBa_K1648049<br> | ||
6. Restriction of BBa_k1172501 with BBa_J13002 in the front, ligase into C backbone and transform into dh5α, forming BBa_K1648050<br> | 6. Restriction of BBa_k1172501 with BBa_J13002 in the front, ligase into C backbone and transform into dh5α, forming BBa_K1648050<br> | ||
Revision as of 18:18, 18 September 2015
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Wet Lab Journal
March
1. Learning Lab techniques and protocols
2. Brainstorming ideas
3. Researches
April
1. Project decided
2. Preparation of special medium and plate for A. vinelandii
3. Study the growth curve characteristics of A. vinelandii
4. Preparation of Competent Cells of dh5α and BL21, aliquot the competent cells, stored in liquid nitrogen and then -80C refrigerator.
5. Test the transformation efficiency of Competent Cells.
6. Make plate with antibiotics -Ampicillin, kanamycin and chloramphenicol.
7. Make LB brooth
May
1. Purchase for primers
2. Primer dilution of the all arrived primers
3. PCR flanking sequence of mamAB operons (main part of BBa_K1648000) from Magnetospirillum gryphiswaldense(MSR-1), following by gel electrophoresis.
4. Restriction of PCR product flanking sequence of mamAB operons, ligase into C backbone and transform into dh5α, forming BBa_K1648000
5. PCR flanking sequence of mamXY, mamGFDC and mms6 operons (main part of BBa_K1648001) from Magnetospirillum gryphiswaldense(MSR-1), following by gel electrophoresis.
6. Restriction of PCR product flanking sequence of mamXY, mamGFDC and mms6 operons, ligase into C backbone and transform into dh5α, forming BBa_K1648001
7. Sending BBa_K1648000 and BBa_K1648001 to sequencing
8. Test the transformation efficiency of Competent Cells
9. Make plate with antibiotics -Ampicillin, kanamycin and chloramphenicol
10. Make LB brooth
June
1. Restriction of BBa_K1648000 with BBa_J13002 in the front and BBa_B0015 at the back, ligase into C backbone and transform into dh5α, forming BBa_K1648002
2. Restriction of BBa_K1648000 with BBa_J13002 in the front and BBa_B0015 at the back, ligase into C backbone and transform into dh5α, forming BBa_K1648003
3. Sending BBa_K1648002 and BBa_K1648003 to sequencing
4. Make LB plate with antibiotics -Ampicillin, kanamycin and chloramphenicol
5. Make LB brooth
July
1. PCR mamHIEJKLMN, mamOPAQRBSTU, mamXY, mamGFDC and mms6 operons from Magnetospirillum gryphiswaldense(MSR-1), following by gel electrophoresis
2. Preparation of Competent Cells of dh5α and BL21, aliquot the competent cells, stored in liquid nitrogen and then -80C refrigerator
3. Make LB plates with antibiotics -Ampicillin, kanamycin and chloramphenicol
4. Make LB brooth
August
1. Preparation of Competent Cells of A. vinelandii
2. Transformation of linearized BBa_K1648002 into A. vinelandii
3. Screening of the transformed A. vinelandii by colony PCR
4. Overlapping PCR of Insertion kit (precursor of BBa_K1648004)
5. Restriction digestion of Overlapping PCR product of Insertion kit, ligase into circular insertion kit and transform into dh5α, forming BBa_K1648004
6. Arrival of synthetic sequence of GFP-nanobody
7. Restriction of synthetic sequence of GFP-nanobody, ligase into C backbone and transform into dh5α, forming BBa_K1648005
8. Restriction of BBa_K1648005, ligase into BBa_K1648004 and transform into dh5α, forming BBa_K1648006
9. Sending BBa_K1648004, BBa_K1648005, BBa_K1648006 to sequencing
10. Mutated oprF is synthesized by IDT and make the PCR product of oprF from Azotobacter vinelandii
11. Make LB, B, BN, BN-MO plates with antibiotics -Ampicillin, kanamycin and chloramphenicol
12. Make LB brooth and BN medium
September
1. Introducing PCR product of mamHIEJKLMN into transformed A. vinelandii
2. Screening of the homologous-recombinant A. vinelandii
3. Restriction of PCR product of oprF and pSB1C3, ligase into C backbone and transform into dh5α, forming BBa_K1648045
4. Restriction of PCR product of oprF with BBa_J13002 in the front, ligase into A backbone and transform into dh5α, forming BBa_K1648048
5. Restriction of synthesized mutated oprF with BBa_R0040 in the front, ligase into C backbone and transform into dh5α, forming BBa_K1648049
6. Restriction of BBa_k1172501 with BBa_J13002 in the front, ligase into C backbone and transform into dh5α, forming BBa_K1648050
7. Make LB, B, BN, BN-MO plates with antibiotics -Ampicillin, kanamycin and chloramphenicol
8. Make LB brooth and BN medium
9. Submission of all biobricks to the registry
