Katarina.94 (Talk | contribs) |
Katarina.94 (Talk | contribs) |
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==Week 1: 13 – 19 June== | ==Week 1: 13 – 19 June== | ||
− | '''14 June''' | + | '''14 June''' |
− | First day at the lab! Making Hutner’s trace elements | + | - First day at the lab! Making Hutner’s trace elements |
Line 15: | Line 15: | ||
'''21 June''' | '''21 June''' | ||
− | Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates. | + | - Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates. ''46 agar plates were made.'' |
− | + | ||
− | + | ||
Line 23: | Line 21: | ||
'''27 June''' | '''27 June''' | ||
− | Transformation of E1010 | + | - Transformation of E1010. ''The transformation was successful.'' |
− | + | ||
− | + | ||
'''28 June''' | '''28 June''' | ||
− | Control of competent cells | + | - Control of competent cells |
'''29 June''' | '''29 June''' | ||
− | Transformation of E1010 to super competent XL-1 | + | - Transformation of E1010 to super competent XL-1. ''The transformation was successful.'' |
− | + | ||
− | + | ||
'''30 June''' | '''30 June''' | ||
− | Making E.Coli Calcium Chloride competent cells | + | - Making E.Coli Calcium Chloride competent cells |
'''1 July''' | '''1 July''' | ||
− | Making solutions for TAP- and TRIS medium | + | - Making solutions for TAP- and TRIS medium. |
− | Cultivation of XL1 and E1010 | + | - Cultivation of XL1 and E1010. |
Line 51: | Line 45: | ||
'''4 July''' | '''4 July''' | ||
− | Making LB-medium and LB-agar. | + | - Making LB-medium and LB-agar. |
− | Plasmid preparation of E1010 | + | - Plasmid preparation of E1010 |
− | Test cultivation of algae | + | - Test cultivation of algae |
'''5 July''' | '''5 July''' | ||
− | Making agar plates | + | - Making agar plates |
− | Digestion and ligation of LIP, U6, UTR and LIP-RFP. | + | - Digestion and ligation of LIP, U6, UTR and LIP-RFP. |
− | Transformation of E1010 and MD-cells competent test | + | - Transformation of E1010 and MD-cells competent test |
− | First algae cultivation | + | - First algae cultivation |
'''6 July''' | '''6 July''' | ||
− | Transformation on U6, LIP, LIP-RFP and UTR. | + | - Transformation on U6, LIP, LIP-RFP and UTR. ''Colonies for U6 and LIP were detected.'' |
− | + | ||
'''7 July''' | '''7 July''' | ||
− | Cultivation of U6, LIP, LIP-RFP colonies on new plates with Chloramphenicol | + | - Cultivation of U6, LIP, LIP-RFP colonies on new plates with Chloramphenicol |
'''8 July''' | '''8 July''' | ||
− | OD measurement of transformated bacteria. | + | - OD measurement of transformated bacteria. |
==Week 5: 11 – 17 July== | ==Week 5: 11 – 17 July== | ||
− | + | '''11 July''' | |
− | + | ||
− | The gel did not show any bands for Cas9 | + | - PCR on Cas9 |
− | + | ||
− | + | '''12 July''' | |
− | + | ||
− | - | + | - Gel electrophoresis on Cas9 to see if the PCR succeeded. ''The gel did not show any bands for Cas9.'' |
− | No bands were obtained on the gel. | + | |
− | - | + | '''13 July''' |
+ | |||
+ | - PCR | ||
+ | |||
+ | '''14 July''' | ||
+ | |||
+ | - PCR on pSB1C3 | ||
+ | |||
+ | '''15 July''' | ||
+ | |||
+ | - Gel electrophoresis on pSB1C3. ''No bands were obtained on the gel.'' | ||
+ | |||
+ | - PCR on pSB1C3 | ||
==Week 6: 18 – 24 July== | ==Week 6: 18 – 24 July== | ||
− | + | '''18 July''' | |
− | No bands were obtained. | + | |
− | + | - PCR and gel electrophoresis on pSB1C3. ''No bands were obtained.'' | |
− | We obtained bands on the gel at approximately 2000 bp. | + | |
− | + | '''20 July''' | |
+ | |||
+ | - PCR and gel electrophoresis on pSB1C3. ''We obtained bands on the gel at approximately 2000 bp.'' | ||
+ | |||
+ | '''22 July''' | ||
+ | |||
+ | - Digestion and ligation on LIP, U6, UTR, Cas9, LIP-RFP, sgRNA and pSB1C3. | ||
==Week 7: 25 – 31 July== | ==Week 7: 25 – 31 July== | ||
− | + | '''25 July''' | |
− | - | + | |
− | - | + | - PCR on LIP and UTR from colonies |
− | - | + | |
− | + | - Cultivation of LIP and UTR colonies on new plates | |
− | - | + | |
− | No bands were obtained. | + | - PCR purification |
− | - | + | |
− | + | '''26 July''' | |
− | Transformation of LIP-RFP and pSB1C3. | + | |
+ | - Gel electrophoresis on pSB1C3, UTR and LIP. ''No bands were obtained.'' | ||
+ | |||
+ | - Digestion and Ligation on LIP-RFP and pSB1C3. | ||
+ | |||
+ | '''27 July''' | ||
+ | |||
+ | - '''''New project approach''''' | ||
+ | |||
+ | - Transformation of LIP-RFP and pSB1C3. | ||
==Week 8: 1 – 7 August== | ==Week 8: 1 – 7 August== | ||
− | + | ||
− | - | + | '''1 August''' |
− | - | + | |
− | Bands were obtained at 700 bp. | + | - Cultivation of Hyg |
− | + | ||
− | - | + | - Gel electrophoresis on UTR and LIP. ''Bands were obtained at 700 bp.'' |
− | Was later show to be wrong | + | |
− | - | + | '''3 August''' |
− | 4 colonies on sgRNA and 4 colonies on LIP-RFP. No colonies on Cas9 and U6. | + | |
− | + | - Plasmid preparation of LIP, UTR and Hyg. ''Was later show to be wrong.'' | |
+ | |||
+ | - Transformation of LIP-RFP, U6, sgRNA and Cas9. ''4 colonies on sgRNA and 4 colonies on LIP-RFP. No colonies on Cas9 and U6.'' | ||
+ | |||
+ | '''4 August''' | ||
+ | |||
+ | - Making TAP medium for cultivation of algae in the dark | ||
==Week 9: 8 – 14 August== | ==Week 9: 8 – 14 August== | ||
− | + | ||
− | - | + | '''8 August''' |
− | - | + | |
− | - | + | - PCR on LIP-RFP and sgRNA |
− | + | ||
− | - | + | - Cultivation of LIP-RFP and sgRNA colonies on new plates |
− | - | + | |
− | No bands on the gel. | + | - First algae cultivation in darkness |
− | + | ||
− | - | + | '''9 August''' |
− | - | + | |
− | Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good. | + | - Transformation of U6 and Cas9. |
− | + | ||
− | - | + | - Gel electrophoresis on LIP-RFP and sgRNA. ''No bands on the gel.'' |
− | - | + | |
− | Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp. | + | '''10 August''' |
− | + | ||
− | Cas9 showed no bands. U6 showed bands at 350 bp. U6 with primers should be at 515 bp. | + | - PCR on LIP-RFP and sgRNA |
+ | |||
+ | - Gel electrophoresis on LIP-RFP. ''Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good.'' | ||
+ | |||
+ | '''11 August''' | ||
+ | |||
+ | - PCR on U6 and Cas9. | ||
+ | |||
+ | - Gel electrophoresis on sgRNA. ''Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp.'' | ||
+ | |||
+ | '''12 August''' | ||
+ | |||
+ | - Gel electrophoresis on Cas9 and U6. ''Cas9 showed no bands. U6 showed bands at 350 bp. U6 with primers should be at 515 bp.'' | ||
==Week 10: 15 – 21 August== | ==Week 10: 15 – 21 August== | ||
− | + | '''15 August''' | |
− | - | + | |
− | Because of difficulties with the gas no plates could be performed today. | + | - Preparation of TAP agar. ''Because of difficulties with the gas no plates could be performed today.'' |
− | - | + | |
− | pSB1C3 showed at 2000 bp as it was supposed to. The other fragments did not show any bands. | + | - Gel electrophoresis on UTR, LIP, Hyg and pSB1C3. ''pSB1C3 showed at 2000 bp as it was supposed to. The other fragments did not show any bands.'' |
− | - | + | |
− | + | - Cultivation of Hyg. | |
− | - | + | |
− | - | + | '''16 August''' |
− | - | + | |
− | The gel showed a weak band on Cas9 around 4000 bp. | + | - Cultivation of sgRNA, LIP-RFP and U6. |
− | + | ||
− | Was later show to be wrong | + | - PCR on Cas9 and Hyg |
− | + | ||
− | - | + | - Gel electrophoresis on Cas9 and Hyg. ''The gel showed a weak band on Cas9 around 4000 bp.'' |
− | It took 5 days for the algae wild type to reach OD 1,757 | + | |
− | The mutant alga evaporated | + | '''17 August''' |
− | - | + | |
− | - | + | - Plasmid preparation on LIP-RFP, U6 and sgRNA. ''Was later show to be wrong.'' |
+ | |||
+ | '''18 August''' | ||
+ | |||
+ | - Cultivation of algae for transformation. ''It took 5 days for the algae wild type to reach OD 1,757. The mutant alga evaporated.'' | ||
+ | |||
+ | - Making TAP agar plates | ||
+ | |||
+ | - Making TAP Hygromycin plates | ||
==Week 11: 22 – 28 August== | ==Week 11: 22 – 28 August== | ||
− | + | ||
− | + | '''22 August''' | |
− | - | + | |
− | - | + | - Cultivation of LIP-RFP and Hyg |
− | - | + | |
− | + | '''23 August''' | |
− | - | + | |
− | - | + | - Plasmid preparation on LIP-RFP and Hyg |
− | Bands for Cas9 and Hyg were obtained. | + | |
− | - | + | - PCR on Cas9 and pSB1C3 |
− | + | ||
− | - | + | - New cultivation of algae in the dark |
− | Bands were detected at 2000 bp which match with pSB1C3 | + | |
− | - | + | '''24 August''' |
− | - | + | |
− | - | + | - PCR on LIP-RFP, Hyg and pSB1C3 |
− | Colonies were obtained! | + | |
− | - | + | - Gel electrophoresis on Cas9, Hyg, LIP-RFP and pSB1C3. ''Bands for Cas9 and Hyg were obtained.'' |
− | + | ||
− | - | + | - PCR purification of Cas9. |
− | - | + | |
− | 52 plates were made | + | '''25 August''' |
− | - | + | |
− | Bands were detected for all the DNAs! | + | - Gel electrophoresis on pSB1C3. ''Bands were detected at 2000 bp which match with pSB1C3'' |
+ | |||
+ | - PCR purification on pSB1C3 | ||
+ | |||
+ | - '''''First Gibson Assembly!''''' | ||
+ | |||
+ | - Transformation of Gibson Assembly. ''Colonies were obtained!'' | ||
+ | |||
+ | - PCR on Cas9, Hyg, pSB1C3 | ||
+ | |||
+ | '''26 August''' | ||
+ | |||
+ | - PCR on Gibson Assembly product and colonies from Gibson Assembly transformation | ||
+ | |||
+ | - Chloramphenicol plates. ''52 plates were made'' | ||
+ | |||
+ | - Gel electrophoresis on PCR product from Gibson Assembly, Cas9, Hyg and pSB1C3. ''Bands were detected for all the DNAs!'' | ||
==Week 12: 29 August – 4 September== | ==Week 12: 29 August – 4 September== | ||
− | + | '''29 August''' | |
− | - | + | |
− | A band at 5500 bp was obtained. We want bands at 7000 bp. | + | - Gel electrophoresis on Gibson Assembly colonies. ''A band at 5500 bp was obtained. We want bands at 7000 bp.'' |
− | - | + | |
− | - | + | - Digestion on LIP-RFP |
− | + | ||
− | - | + | - PCR on Gibson Assembly colonies. |
− | - | + | |
− | - | + | '''30 August''' |
− | - | + | |
− | + | - PCR on Gibson Assembly colonies. | |
− | - | + | |
− | No bands. | + | - Cultivation of Gibson Assembly coloni on new plates |
− | - | + | |
− | + | - Ligation on LIP-RFP with pSB1C3. | |
− | - | + | |
− | - | + | - New Gibson Assembly transformation |
− | No result on the gel. | + | |
− | + | '''31 August''' | |
− | - | + | |
− | - | + | - Gel electrophoresis on Gibson Assembly colonies. ''No bands.'' |
− | - | + | |
− | + | - Plasmid preparation on Gibson Assembly colony. | |
− | - PCR and gel electrophoresis on gibson colonies | + | |
− | No results | + | '''1 September''' |
+ | |||
+ | - PCR on plasmid prepared U6, UTR, LIP, LIP-RFP, sgRNA and Hyg. | ||
+ | |||
+ | - Gel electrophoresis on LIP, sgRNA, U6, UTR, LIP-RFP and Hyg. ''No result on the gel.'' | ||
+ | |||
+ | '''2 September''' | ||
+ | |||
+ | - Second Gibson assembly | ||
+ | |||
+ | - Gibson transformation | ||
+ | |||
+ | - Transformation LIP-RFP | ||
+ | |||
+ | '''3 September''' | ||
+ | |||
+ | - PCR and gel electrophoresis on gibson colonies. ''No results'' | ||
+ | |||
- Digestion and ligation of LIP, U6, UTR, sgRNA and pSB1C3 | - Digestion and ligation of LIP, U6, UTR, sgRNA and pSB1C3 | ||
− | + | ||
− | - PCR and gel electrophoresis on gibson colonies | + | '''4 September''' |
− | It looks like Colony 8 has a band at 7000 bp! Yeeey! | + | |
+ | - PCR and gel electrophoresis on gibson colonies. ''It looks like Colony 8 has a band at 7000 bp! Yeeey!'' | ||
==Week 13: 5 – 11 September== | ==Week 13: 5 – 11 September== | ||
− | + | '''5 September''' | |
− | PCR on old colonies of LIP-RFP | + | |
− | Making LB-medium | + | - PCR on old colonies of LIP-RFP |
− | Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies. | + | |
− | + | - Making LB-medium | |
− | Cultivation of colony 8 (Gibson Assembly) with Hyg in the LB-media | + | |
− | Screening of colonies from Gibson Assembly | + | - Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies. |
− | Bands were obtained, but no band was at 7000 bp. | + | |
− | + | '''6 September''' | |
− | PCR and gel electrophoresis on Hyg | + | |
− | + | - Cultivation of colony 8 (Gibson Assembly) with Hyg in the LB-media | |
− | Plasmid preparation of Gibson Assembly colony 8 | + | |
− | Screening on Gibson colonies | + | - Screening of colonies from Gibson Assembly. ''Bands were obtained, but no band was at 7000 bp.'' |
− | + | ||
− | The sequences were obtained | + | '''7 September''' |
− | We did not insert Hyg but instead YFP was inserted. | + | |
− | Cas9 and LIP are inserted successfully! | + | - PCR and gel electrophoresis on Hyg |
− | + | ||
− | Cultivation of algae mutants and Gibson colony 8 | + | '''8 September''' |
− | Gel electrophoresis on plasmid preparation of Gibson colony 8 and Gibson colonies | + | |
− | The plasmid preparation of Gibson colony 8 showed good bands. | + | - Plasmid preparation of Gibson Assembly colony 8 |
− | + | ||
− | PCR on some Gibson colonies | + | - Screening on Gibson colonies |
− | Preparation for plasmid preparation | + | |
− | The cells were centrifuged and the pellet was saved for later continuation of plasmid preparation | + | '''9 September''' |
+ | - '''''The sequences were obtained''''' ''We did not insert Hyg but instead YFP was inserted. Cas9 and LIP are inserted successfully!'' | ||
+ | |||
+ | '''10 September''' | ||
+ | |||
+ | - Cultivation of algae mutants and Gibson colony 8 | ||
+ | |||
+ | - Gel electrophoresis on plasmid preparation of Gibson colony 8 and Gibson colonies. ''The plasmid preparation of Gibson colony 8 showed good bands.'' | ||
+ | |||
+ | '''11 September''' | ||
+ | |||
+ | - PCR on some Gibson colonies | ||
+ | |||
+ | - Preparation for plasmid preparation. The cells were centrifuged and the pellet was saved for later continuation of plasmid preparation | ||
Cultivation of Gibson Assembly colonies on new plates | Cultivation of Gibson Assembly colonies on new plates | ||
==Week 14: 12 – 18 September== | ==Week 14: 12 – 18 September== | ||
− | + | '''13 September''' | |
− | OD measurments on the algae | + | |
− | Gel electrophoresis on the PCR product from 11/9 - 16. | + | - OD measurments on the algae |
− | Band on 300 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp were obtained. | + | |
− | + | - Gel electrophoresis on the PCR product from 11/9 - 16. ''Band on 300 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp were obtained.'' | |
− | Dilution of the algae | + | |
− | Making TAP-40mM sucrose | + | '''14 September''' |
− | Plasmid preparation of Gibson Assembly colony 8 | + | |
− | + | - Dilution of the algae | |
− | Digestion of Gibson colony 8 | + | |
− | + | - Making TAP-40mM sucrose | |
− | Electroporation on algae | + | |
− | The algae have grown well. | + | - Plasmid preparation of Gibson Assembly colony 8 |
− | + | ||
− | PCR on Gibson colonies | + | '''15 September''' |
− | Continuation on the electroporation from previous day. | + | |
− | + | - Digestion of Gibson colony 8 | |
− | Gel electrophoresis on Gibson colonies | + | |
+ | '''16 September''' | ||
+ | |||
+ | - Electroporation on algae ''The algae have grown well.'' | ||
+ | |||
+ | '''17 September''' | ||
+ | |||
+ | - PCR on Gibson colonies | ||
+ | |||
+ | - Continuation on the electroporation from previous day. | ||
+ | |||
+ | '''18 September''' | ||
+ | |||
+ | - Gel electrophoresis on Gibson colonies | ||
==Week 15: 19 – 25 September== | ==Week 15: 19 – 25 September== | ||
− | + | '''19 September''' | |
− | Sequenced was obtained | + | |
− | Looks like we did not insert U6 and sgRNA :( | + | - Sequenced was obtained. ''Looks like we did not insert U6 and sgRNA :('' |
− | + | ||
− | PCR on Gibson 3 | + | '''21 September''' |
− | Gel electrophoresis on the PCR product from today | + | |
− | Band were obtained at 300 bp and 2000 bp. | + | - PCR on Gibson 3 |
− | + | ||
− | Gel electrophoresis on PCR product from yesterday | + | - Gel electrophoresis on the PCR product from today. ''Band were obtained at 300 bp and 2000 bp.'' |
− | Bands at 2000 bp and 300 bp were obtained | + | |
− | Gibson on pSB1C3 with LIP, LIP-RFP, U6 and UTR respectively. | + | '''22 September''' |
− | Transformation on all the Gibson product | + | |
− | + | - Gel electrophoresis on PCR product from yesterday. ''Bands at 2000 bp and 300 bp were obtained'' | |
− | Gel electrophoresis on Gibson 3 colonies | + | |
− | No bands | + | - Gibson on pSB1C3 with LIP, LIP-RFP, U6 and UTR respectively. |
− | PCR of Gibson with LIP, LIP-RFP, U6 and Term. | + | |
− | Screening of YFP transformed algae | + | - Transformation on all the Gibson product |
− | No proof that the transformation worked | + | |
− | + | '''23 September''' | |
− | Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP | + | |
− | Bands were obtained at 500 bp for Term, 600 bp for LIP and 1500 bp for LIP-RFP. No result for U6 | + | - Gel electrophoresis on Gibson 3 colonies. ''No bands'' |
− | Gel electrophoresis on Gibson 3 colonies | + | |
− | No bands | + | - PCR of Gibson with LIP, LIP-RFP, U6 and Term. |
− | PCR on Gibson with U6, Term, LIP and LIP-RFP | + | |
− | Cultivation of U6, Term, LIP and LIP-RFP | + | - Screening of YFP transformed algae. ''No proof that the transformation worked'' |
− | + | ||
− | PCR on Gibson 3 colonies | + | '''24 September''' |
− | Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. | + | - Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. ''Bands were obtained at 500 bp for Term, 600 bp for LIP and 1500 bp for LIP-RFP. No result for U6'' |
− | Bands were obatined | + | |
− | Cultivation of U6, Term, LIP and LIP-RFP | + | - Gel electrophoresis on Gibson 3 colonies. ''No bands'' |
+ | |||
+ | - PCR on Gibson with U6, Term, LIP and LIP-RFP | ||
+ | |||
+ | - Cultivation of U6, Term, LIP and LIP-RFP | ||
+ | |||
+ | '''25 September''' | ||
+ | |||
+ | - PCR on Gibson 3 colonies | ||
+ | |||
+ | - Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. ''Bands were obatined'' | ||
+ | |||
+ | - Cultivation of U6, Term, LIP and LIP-RFP | ||
==Week 16: 26 September - 2 October== | ==Week 16: 26 September - 2 October== | ||
− | + | '''26 September''' | |
− | PCR on U6 colonies | + | |
− | Gel electrophoresis on Gibson 3 colonies | + | - PCR on U6 colonies |
− | No result | + | |
− | Plasmid preparation on LIP, LIP-RFP and Term. | + | - Gel electrophoresis on Gibson 3 colonies. ''No result'' |
− | + | ||
− | Plasmid preparation nr 2 on LIP, LIP-RFP and Term. | + | - Plasmid preparation on LIP, LIP-RFP and Term. |
− | Gel electrophoresis on U6 colonies | + | |
− | No result | + | '''27 September''' |
− | PCR on Gibson 3 colonies | + | |
− | + | - Plasmid preparation nr 2 on LIP, LIP-RFP and Term. | |
− | Gel electrophoresis on Gibson 3 colonies | + | |
− | No result | + | - Gel electrophoresis on U6 colonies. ''No result'' |
− | Cultivation of U6, Term, LIP and LIP-RFP | + | |
− | PCR on Gibson 3 colonies. | + | - PCR on Gibson 3 colonies |
− | + | ||
− | Gel electrophoresis | + | '''28 September''' |
− | + | ||
− | Gel electrophoresis on LIP, LIP-RFP and Terminator | + | - Gel electrophoresis on Gibson 3 colonies. ''No result'' |
− | Bands were obtained | + | |
− | Plasmid preparation on LIP, LIP-RFP and Terminator | + | - Cultivation of U6, Term, LIP and LIP-RFP |
− | New cultivation of LIP on plates | + | |
− | + | - PCR on Gibson 3 colonies. | |
− | Gel electrophoresis on LIP, LIP-RFP and Terminator | + | |
− | Bands were obtained | + | '''29 September''' |
+ | |||
+ | - Gel electrophoresis | ||
+ | |||
+ | '''1 October''' | ||
+ | |||
+ | - Gel electrophoresis on LIP, LIP-RFP and Terminator. ''Bands were obtained'' | ||
+ | |||
+ | - Plasmid preparation on LIP, LIP-RFP and Terminator | ||
+ | |||
+ | - New cultivation of LIP on plates | ||
+ | |||
+ | '''2 October''' | ||
+ | |||
+ | - Gel electrophoresis on LIP, LIP-RFP and Terminator. ''Bands were obtained'' | ||
==Week 17: 3 - 9 October== | ==Week 17: 3 - 9 October== | ||
− | + | '''3 October''' | |
− | Sequencing of LIP, LIP-RFP and Term | + | |
− | + | - Sequencing of LIP, LIP-RFP and Term | |
− | Gibson Assembly on U6 | + | |
− | Transformation on U6 | + | '''5 October''' |
− | + | ||
− | PCR on U6 | + | - Gibson Assembly on U6 |
− | Cultivation of LIP-RFP | + | |
− | + | - Transformation on U6 | |
− | Gel electrophoresis on U6 | + | |
− | No insert | + | '''6 October''' |
− | Cultivation of algae | + | |
− | Cultivation of LIP-RFP | + | - PCR on U6 |
− | + | ||
− | Cultivation of LIP-RFP | + | - Cultivation of LIP-RFP |
− | + | ||
− | Plasmid preparation on LIP-RFP | + | '''7 October''' |
+ | |||
+ | - Gel electrophoresis on U6. ''No insert'' | ||
+ | |||
+ | - Cultivation of algae | ||
+ | |||
+ | - Cultivation of LIP-RFP | ||
+ | |||
+ | '''8 October''' | ||
+ | |||
+ | - Cultivation of LIP-RFP | ||
+ | |||
+ | '''9 October''' | ||
+ | |||
+ | - Plasmid preparation on LIP-RFP | ||
{{Linkoping_Sweden/Footer}} | {{Linkoping_Sweden/Footer}} |
Revision as of 12:37, 11 October 2016
Contents
- 1 Experiments - Overview on Laboration
- 1.1 Week 1: 13 – 19 June
- 1.2 Week 2: 20 – 26 June
- 1.3 Week 3: 27 June – 3 July
- 1.4 Week 4: 4 – 10 July
- 1.5 Week 5: 11 – 17 July
- 1.6 Week 6: 18 – 24 July
- 1.7 Week 7: 25 – 31 July
- 1.8 Week 8: 1 – 7 August
- 1.9 Week 9: 8 – 14 August
- 1.10 Week 10: 15 – 21 August
- 1.11 Week 11: 22 – 28 August
- 1.12 Week 12: 29 August – 4 September
- 1.13 Week 13: 5 – 11 September
- 1.14 Week 14: 12 – 18 September
- 1.15 Week 15: 19 – 25 September
- 1.16 Week 16: 26 September - 2 October
- 1.17 Week 17: 3 - 9 October
Experiments - Overview on Laboration
Week 1: 13 – 19 June
14 June
- First day at the lab! Making Hutner’s trace elements
Week 2: 20 – 26 June
21 June
- Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates. 46 agar plates were made.
Week 3: 27 June – 3 July
27 June
- Transformation of E1010. The transformation was successful.
28 June
- Control of competent cells
29 June
- Transformation of E1010 to super competent XL-1. The transformation was successful.
30 June
- Making E.Coli Calcium Chloride competent cells
1 July
- Making solutions for TAP- and TRIS medium.
- Cultivation of XL1 and E1010.
Week 4: 4 – 10 July
4 July
- Making LB-medium and LB-agar.
- Plasmid preparation of E1010
- Test cultivation of algae
5 July
- Making agar plates
- Digestion and ligation of LIP, U6, UTR and LIP-RFP.
- Transformation of E1010 and MD-cells competent test
- First algae cultivation
6 July
- Transformation on U6, LIP, LIP-RFP and UTR. Colonies for U6 and LIP were detected.
7 July
- Cultivation of U6, LIP, LIP-RFP colonies on new plates with Chloramphenicol
8 July
- OD measurement of transformated bacteria.
Week 5: 11 – 17 July
11 July
- PCR on Cas9
12 July
- Gel electrophoresis on Cas9 to see if the PCR succeeded. The gel did not show any bands for Cas9.
13 July
- PCR
14 July
- PCR on pSB1C3
15 July
- Gel electrophoresis on pSB1C3. No bands were obtained on the gel.
- PCR on pSB1C3
Week 6: 18 – 24 July
18 July
- PCR and gel electrophoresis on pSB1C3. No bands were obtained.
20 July
- PCR and gel electrophoresis on pSB1C3. We obtained bands on the gel at approximately 2000 bp.
22 July
- Digestion and ligation on LIP, U6, UTR, Cas9, LIP-RFP, sgRNA and pSB1C3.
Week 7: 25 – 31 July
25 July
- PCR on LIP and UTR from colonies
- Cultivation of LIP and UTR colonies on new plates
- PCR purification
26 July
- Gel electrophoresis on pSB1C3, UTR and LIP. No bands were obtained.
- Digestion and Ligation on LIP-RFP and pSB1C3.
27 July
- New project approach
- Transformation of LIP-RFP and pSB1C3.
Week 8: 1 – 7 August
1 August
- Cultivation of Hyg
- Gel electrophoresis on UTR and LIP. Bands were obtained at 700 bp.
3 August
- Plasmid preparation of LIP, UTR and Hyg. Was later show to be wrong.
- Transformation of LIP-RFP, U6, sgRNA and Cas9. 4 colonies on sgRNA and 4 colonies on LIP-RFP. No colonies on Cas9 and U6.
4 August
- Making TAP medium for cultivation of algae in the dark
Week 9: 8 – 14 August
8 August
- PCR on LIP-RFP and sgRNA
- Cultivation of LIP-RFP and sgRNA colonies on new plates
- First algae cultivation in darkness
9 August
- Transformation of U6 and Cas9.
- Gel electrophoresis on LIP-RFP and sgRNA. No bands on the gel.
10 August
- PCR on LIP-RFP and sgRNA
- Gel electrophoresis on LIP-RFP. Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good.
11 August
- PCR on U6 and Cas9.
- Gel electrophoresis on sgRNA. Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp.
12 August
- Gel electrophoresis on Cas9 and U6. Cas9 showed no bands. U6 showed bands at 350 bp. U6 with primers should be at 515 bp.
Week 10: 15 – 21 August
15 August
- Preparation of TAP agar. Because of difficulties with the gas no plates could be performed today.
- Gel electrophoresis on UTR, LIP, Hyg and pSB1C3. pSB1C3 showed at 2000 bp as it was supposed to. The other fragments did not show any bands.
- Cultivation of Hyg.
16 August
- Cultivation of sgRNA, LIP-RFP and U6.
- PCR on Cas9 and Hyg
- Gel electrophoresis on Cas9 and Hyg. The gel showed a weak band on Cas9 around 4000 bp.
17 August
- Plasmid preparation on LIP-RFP, U6 and sgRNA. Was later show to be wrong.
18 August
- Cultivation of algae for transformation. It took 5 days for the algae wild type to reach OD 1,757. The mutant alga evaporated.
- Making TAP agar plates
- Making TAP Hygromycin plates
Week 11: 22 – 28 August
22 August
- Cultivation of LIP-RFP and Hyg
23 August
- Plasmid preparation on LIP-RFP and Hyg
- PCR on Cas9 and pSB1C3
- New cultivation of algae in the dark
24 August
- PCR on LIP-RFP, Hyg and pSB1C3
- Gel electrophoresis on Cas9, Hyg, LIP-RFP and pSB1C3. Bands for Cas9 and Hyg were obtained.
- PCR purification of Cas9.
25 August
- Gel electrophoresis on pSB1C3. Bands were detected at 2000 bp which match with pSB1C3
- PCR purification on pSB1C3
- First Gibson Assembly!
- Transformation of Gibson Assembly. Colonies were obtained!
- PCR on Cas9, Hyg, pSB1C3
26 August
- PCR on Gibson Assembly product and colonies from Gibson Assembly transformation
- Chloramphenicol plates. 52 plates were made
- Gel electrophoresis on PCR product from Gibson Assembly, Cas9, Hyg and pSB1C3. Bands were detected for all the DNAs!
Week 12: 29 August – 4 September
29 August
- Gel electrophoresis on Gibson Assembly colonies. A band at 5500 bp was obtained. We want bands at 7000 bp.
- Digestion on LIP-RFP
- PCR on Gibson Assembly colonies.
30 August
- PCR on Gibson Assembly colonies.
- Cultivation of Gibson Assembly coloni on new plates
- Ligation on LIP-RFP with pSB1C3.
- New Gibson Assembly transformation
31 August
- Gel electrophoresis on Gibson Assembly colonies. No bands.
- Plasmid preparation on Gibson Assembly colony.
1 September
- PCR on plasmid prepared U6, UTR, LIP, LIP-RFP, sgRNA and Hyg.
- Gel electrophoresis on LIP, sgRNA, U6, UTR, LIP-RFP and Hyg. No result on the gel.
2 September
- Second Gibson assembly
- Gibson transformation
- Transformation LIP-RFP
3 September
- PCR and gel electrophoresis on gibson colonies. No results
- Digestion and ligation of LIP, U6, UTR, sgRNA and pSB1C3
4 September
- PCR and gel electrophoresis on gibson colonies. It looks like Colony 8 has a band at 7000 bp! Yeeey!
Week 13: 5 – 11 September
5 September
- PCR on old colonies of LIP-RFP
- Making LB-medium
- Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies.
6 September
- Cultivation of colony 8 (Gibson Assembly) with Hyg in the LB-media
- Screening of colonies from Gibson Assembly. Bands were obtained, but no band was at 7000 bp.
7 September
- PCR and gel electrophoresis on Hyg
8 September
- Plasmid preparation of Gibson Assembly colony 8
- Screening on Gibson colonies
9 September - The sequences were obtained We did not insert Hyg but instead YFP was inserted. Cas9 and LIP are inserted successfully!
10 September
- Cultivation of algae mutants and Gibson colony 8
- Gel electrophoresis on plasmid preparation of Gibson colony 8 and Gibson colonies. The plasmid preparation of Gibson colony 8 showed good bands.
11 September
- PCR on some Gibson colonies
- Preparation for plasmid preparation. The cells were centrifuged and the pellet was saved for later continuation of plasmid preparation Cultivation of Gibson Assembly colonies on new plates
Week 14: 12 – 18 September
13 September
- OD measurments on the algae
- Gel electrophoresis on the PCR product from 11/9 - 16. Band on 300 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp were obtained.
14 September
- Dilution of the algae
- Making TAP-40mM sucrose
- Plasmid preparation of Gibson Assembly colony 8
15 September
- Digestion of Gibson colony 8
16 September
- Electroporation on algae The algae have grown well.
17 September
- PCR on Gibson colonies
- Continuation on the electroporation from previous day.
18 September
- Gel electrophoresis on Gibson colonies
Week 15: 19 – 25 September
19 September
- Sequenced was obtained. Looks like we did not insert U6 and sgRNA :(
21 September
- PCR on Gibson 3
- Gel electrophoresis on the PCR product from today. Band were obtained at 300 bp and 2000 bp.
22 September
- Gel electrophoresis on PCR product from yesterday. Bands at 2000 bp and 300 bp were obtained
- Gibson on pSB1C3 with LIP, LIP-RFP, U6 and UTR respectively.
- Transformation on all the Gibson product
23 September
- Gel electrophoresis on Gibson 3 colonies. No bands
- PCR of Gibson with LIP, LIP-RFP, U6 and Term.
- Screening of YFP transformed algae. No proof that the transformation worked
24 September - Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. Bands were obtained at 500 bp for Term, 600 bp for LIP and 1500 bp for LIP-RFP. No result for U6
- Gel electrophoresis on Gibson 3 colonies. No bands
- PCR on Gibson with U6, Term, LIP and LIP-RFP
- Cultivation of U6, Term, LIP and LIP-RFP
25 September
- PCR on Gibson 3 colonies
- Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. Bands were obatined
- Cultivation of U6, Term, LIP and LIP-RFP
Week 16: 26 September - 2 October
26 September
- PCR on U6 colonies
- Gel electrophoresis on Gibson 3 colonies. No result
- Plasmid preparation on LIP, LIP-RFP and Term.
27 September
- Plasmid preparation nr 2 on LIP, LIP-RFP and Term.
- Gel electrophoresis on U6 colonies. No result
- PCR on Gibson 3 colonies
28 September
- Gel electrophoresis on Gibson 3 colonies. No result
- Cultivation of U6, Term, LIP and LIP-RFP
- PCR on Gibson 3 colonies.
29 September
- Gel electrophoresis
1 October
- Gel electrophoresis on LIP, LIP-RFP and Terminator. Bands were obtained
- Plasmid preparation on LIP, LIP-RFP and Terminator
- New cultivation of LIP on plates
2 October
- Gel electrophoresis on LIP, LIP-RFP and Terminator. Bands were obtained
Week 17: 3 - 9 October
3 October
- Sequencing of LIP, LIP-RFP and Term
5 October
- Gibson Assembly on U6
- Transformation on U6
6 October
- PCR on U6
- Cultivation of LIP-RFP
7 October
- Gel electrophoresis on U6. No insert
- Cultivation of algae
- Cultivation of LIP-RFP
8 October
- Cultivation of LIP-RFP
9 October
- Plasmid preparation on LIP-RFP