Difference between revisions of "Team:Linkoping Sweden/Experiments"
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'''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. |
| Line 21: | Line 21: | ||
'''27 June''' | '''27 June''' | ||
| − | - Transformation of E1010. ''The transformation was successful.'' | + | - Transformation of E1010. ''Observation: 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. ''The transformation was successful.'' | + | - Transformation of E1010 to super competent XL-1. ''Observation: 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''' | ||
| Line 47: | Line 47: | ||
- 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. ''Colonies for U6 and LIP were detected.'' | + | - Transformation on U6, LIP, LIP-RFP and UTR. ''Observation: 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''' | ||
| Line 77: | Line 77: | ||
'''11 July''' | '''11 July''' | ||
| − | - PCR on Cas9 | + | - PCR on Cas9. |
'''12 July''' | '''12 July''' | ||
| − | - Gel electrophoresis on Cas9 to see if the PCR | + | - Gel electrophoresis on Cas9 to see if the PCR was successful. '''Observation: No bands were obtained for Cas9.'' |
'''13 July''' | '''13 July''' | ||
| Line 89: | Line 89: | ||
'''14 July''' | '''14 July''' | ||
| − | - PCR on pSB1C3 | + | - PCR on pSB1C3. |
'''15 July''' | '''15 July''' | ||
| − | - Gel electrophoresis on pSB1C3. ''No bands were obtained on the gel.'' | + | - Gel electrophoresis on pSB1C3. ''Observation: No bands were obtained on the gel.'' |
| − | - PCR on pSB1C3 | + | - PCR on pSB1C3. |
| Line 101: | Line 101: | ||
'''18 July''' | '''18 July''' | ||
| − | - PCR and gel electrophoresis on pSB1C3. ''No bands were obtained.'' | + | - PCR and gel electrophoresis on pSB1C3. ''Observation: No bands were obtained on the gel.'' |
'''20 July''' | '''20 July''' | ||
| − | - PCR and gel electrophoresis on pSB1C3. '' | + | - PCR and gel electrophoresis on pSB1C3. ''Observation: Bands were obtained on the gel at approximately 2000 bp.'' |
'''22 July''' | '''22 July''' | ||
| Line 115: | Line 115: | ||
'''25 July''' | '''25 July''' | ||
| − | - PCR on LIP and UTR from colonies | + | - PCR on LIP and UTR from colonies. |
| − | - Cultivation of LIP and UTR colonies on new plates | + | - Cultivation of LIP and UTR colonies on new plates. |
| − | - PCR purification | + | - PCR purification. |
'''26 July''' | '''26 July''' | ||
| − | - Gel electrophoresis on pSB1C3, UTR and LIP. ''No bands were obtained.'' | + | - Gel electrophoresis on pSB1C3, UTR and LIP. ''Observation: No bands were obtained on the gel.'' |
- Digestion and Ligation on LIP-RFP and pSB1C3. | - Digestion and Ligation on LIP-RFP and pSB1C3. | ||
| Line 138: | Line 138: | ||
'''1 August''' | '''1 August''' | ||
| − | - Cultivation of | + | - Cultivation of Hygromycin. |
| − | - Gel electrophoresis on UTR and LIP. ''Bands were obtained at 700 bp.'' | + | - Gel electrophoresis on UTR and LIP. ''Observation: Bands were obtained on the gel at 700 bp.'' |
'''3 August''' | '''3 August''' | ||
| − | - Plasmid preparation of LIP, UTR and Hyg. '' | + | - Plasmid preparation of LIP, UTR and Hyg. ''Observation: Turned out to be incorrect later on.'' |
| − | - Transformation of LIP-RFP, U6, sgRNA and Cas9. ''4 colonies on sgRNA and 4 colonies on LIP-RFP. No colonies on Cas9 and U6.'' | + | - Transformation of LIP-RFP, U6, sgRNA and Cas9. ''Observation: 4 colonies on sgRNA and 4 colonies on LIP-RFP. No colonies on Cas9 and U6.'' |
'''4 August''' | '''4 August''' | ||
| − | - Making TAP medium for cultivation of algae in the dark | + | - Making TAP medium for cultivation of algae in the dark. |
| Line 157: | Line 157: | ||
'''8 August''' | '''8 August''' | ||
| − | - PCR on LIP-RFP and sgRNA | + | - PCR on LIP-RFP and sgRNA. |
| − | - Cultivation of LIP-RFP and sgRNA colonies on new plates | + | - Cultivation of LIP-RFP and sgRNA colonies on new plates. |
| − | - First algae cultivation in darkness | + | - First algae cultivation in darkness. |
'''9 August''' | '''9 August''' | ||
| Line 167: | Line 167: | ||
- Transformation of U6 and Cas9. | - Transformation of U6 and Cas9. | ||
| − | - Gel electrophoresis on LIP-RFP and sgRNA. ''No bands on the gel.'' | + | - Gel electrophoresis on LIP-RFP and sgRNA. ''Observation: No bands on the gel.'' |
'''10 August''' | '''10 August''' | ||
| Line 173: | Line 173: | ||
- PCR on LIP-RFP and sgRNA | - 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.'' | + | - Gel electrophoresis on LIP-RFP. ''Observation: Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good.'' |
'''11 August''' | '''11 August''' | ||
| Line 179: | Line 179: | ||
- PCR on U6 and Cas9. | - PCR on U6 and Cas9. | ||
| − | - Gel electrophoresis on sgRNA. ''Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp.'' | + | - Gel electrophoresis on sgRNA. ''Observation: Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp.'' |
'''12 August''' | '''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.'' | + | - Gel electrophoresis on Cas9 and U6. ''Observation: Cas9 showed no bands. U6 showed bands at 350 bp. U6 with primers should be at 515 bp.'' |
| Line 189: | Line 189: | ||
'''15 August''' | '''15 August''' | ||
| − | - Preparation of TAP agar. ''Because of difficulties with the gas no plates could be performed today.'' | + | - Preparation of TAP agar. ''Observation: 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.'' | + | - Gel electrophoresis on UTR, LIP, Hyg and pSB1C3. ''pSB1C3 showed a band at 2000 bp as it was supposed to. The other fragments did not show any bands.'' |
- Cultivation of Hyg. | - Cultivation of Hyg. | ||
| Line 199: | Line 199: | ||
- Cultivation of sgRNA, LIP-RFP and U6. | - Cultivation of sgRNA, LIP-RFP and U6. | ||
| − | - PCR on Cas9 and Hyg | + | - PCR on Cas9 and Hyg. |
| − | - Gel electrophoresis on Cas9 and Hyg. ''The gel showed a weak band on Cas9 around 4000 bp.'' | + | - Gel electrophoresis on Cas9 and Hyg. ''Observation: The gel showed a weak band on Cas9 around 4000 bp.'' |
'''17 August''' | '''17 August''' | ||
| − | - Plasmid preparation on LIP-RFP, U6 and sgRNA. '' | + | - Plasmid preparation on LIP-RFP, U6 and sgRNA. '''Observation: Turned out to be incorrect later on.'' |
'''18 August''' | '''18 August''' | ||
| − | - Cultivation of algae for transformation. ''It took 5 days for the algae wild type to reach OD 1,757. The mutant | + | - Cultivation of algae for transformation. '''Observation: It took 5 days for the algae wild type to reach OD = 1,757. The mutant algae evaporated.'' |
| − | - Making TAP agar plates | + | - Making TAP agar plates. |
| − | - Making TAP | + | - Making TAP Hyg. plates. |
| Line 220: | Line 220: | ||
'''22 August''' | '''22 August''' | ||
| − | - Cultivation of LIP-RFP and Hyg | + | - Cultivation of LIP-RFP and Hyg. |
'''23 August''' | '''23 August''' | ||
| − | - Plasmid preparation on LIP-RFP and Hyg | + | - Plasmid preparation on LIP-RFP and Hyg. |
| − | - PCR on Cas9 and pSB1C3 | + | - PCR on Cas9 and pSB1C3. |
| − | - New cultivation of algae in the dark | + | - New cultivation of algae in the dark. |
'''24 August''' | '''24 August''' | ||
| − | - PCR on LIP-RFP, Hyg and pSB1C3 | + | - PCR on LIP-RFP, Hyg and pSB1C3. |
| − | - Gel electrophoresis on Cas9, Hyg, LIP-RFP and pSB1C3. ''Bands for Cas9 and Hyg were obtained.'' | + | - Gel electrophoresis on Cas9, Hyg, LIP-RFP and pSB1C3. ''Observation: Bands for Cas9 and Hyg were obtained.'' |
- PCR purification of Cas9. | - PCR purification of Cas9. | ||
| Line 240: | Line 240: | ||
'''25 August''' | '''25 August''' | ||
| − | - Gel electrophoresis on pSB1C3. ''Bands were detected at 2000 bp which | + | - Gel electrophoresis on pSB1C3. ''Observation: Bands were detected at 2000 bp which corresponds to the size of pSB1C3'' |
| − | - PCR purification on pSB1C3 | + | - PCR purification on pSB1C3. |
- '''''First Gibson Assembly!''''' | - '''''First Gibson Assembly!''''' | ||
| − | - Transformation of Gibson Assembly. ''Colonies were obtained!'' | + | - Transformation of Gibson Assembly. ''Observation: Colonies were obtained!'' |
| − | - PCR on Cas9, Hyg, pSB1C3 | + | - PCR on Cas9, Hyg, pSB1C3. |
'''26 August''' | '''26 August''' | ||
| − | - PCR on Gibson Assembly product and colonies from Gibson Assembly transformation | + | - PCR on Gibson Assembly product and colonies from Gibson Assembly transformation. |
| − | - Chloramphenicol plates. | + | - Chloramphenicol plates. |
| − | - Gel electrophoresis on PCR product from Gibson Assembly, Cas9, Hyg and pSB1C3. ''Bands were detected for all the DNAs!'' | + | - Gel electrophoresis on PCR product from Gibson Assembly, Cas9, Hyg and pSB1C3. ''Observation: Bands were detected for all the DNAs!'' |
| Line 262: | Line 262: | ||
'''29 August''' | '''29 August''' | ||
| − | - Gel electrophoresis on Gibson Assembly colonies. ''A band at 5500 bp was obtained. We want bands at 7000 bp.'' | + | - Gel electrophoresis on Gibson Assembly colonies. ''Observation: A band at 5500 bp was obtained. We want bands at 7000 bp.'' |
| − | - Digestion on LIP-RFP | + | - Digestion on LIP-RFP. |
- PCR on Gibson Assembly colonies. | - PCR on Gibson Assembly colonies. | ||
| Line 272: | Line 272: | ||
- PCR on Gibson Assembly colonies. | - PCR on Gibson Assembly colonies. | ||
| − | - Cultivation of Gibson Assembly | + | - Cultivation of Gibson Assembly colonies on new plates. |
- Ligation on LIP-RFP with pSB1C3. | - Ligation on LIP-RFP with pSB1C3. | ||
| − | - New Gibson Assembly transformation | + | - New Gibson Assembly transformation. |
'''31 August''' | '''31 August''' | ||
| − | - Gel electrophoresis on Gibson Assembly colonies. ''No bands.'' | + | - Gel electrophoresis on Gibson Assembly colonies. ''Observation: No bands on the gel.'' |
- Plasmid preparation on Gibson Assembly colony. | - Plasmid preparation on Gibson Assembly colony. | ||
| Line 288: | Line 288: | ||
- PCR on plasmid prepared U6, UTR, LIP, LIP-RFP, sgRNA and Hyg. | - PCR on plasmid prepared U6, UTR, LIP, LIP-RFP, sgRNA and Hyg. | ||
| − | - Gel electrophoresis on LIP, sgRNA, U6, UTR, LIP-RFP and Hyg. ''No | + | - Gel electrophoresis on LIP, sgRNA, U6, UTR, LIP-RFP and Hyg. ''Observation: No bands on the gel.'' |
'''2 September''' | '''2 September''' | ||
| − | - Second Gibson assembly | + | - Second Gibson assembly. |
| − | - Gibson transformation | + | - Gibson transformation. |
| − | - Transformation LIP-RFP | + | - Transformation of LIP-RFP. |
'''3 September''' | '''3 September''' | ||
| − | - PCR and gel electrophoresis on gibson colonies. ''No results'' | + | - PCR and gel electrophoresis on gibson colonies. ''Observation: No results'' |
| − | - Digestion and ligation of LIP, U6, UTR, sgRNA and pSB1C3 | + | - Digestion and ligation of LIP, U6, UTR, sgRNA and pSB1C3. |
'''4 September''' | '''4 September''' | ||
| − | - PCR and gel electrophoresis on gibson colonies. ''It looks like Colony 8 has a band at 7000 bp! Yeeey!'' | + | - PCR and gel electrophoresis on gibson colonies. ''Observation: It looks like Colony 8 has a band at 7000 bp! Yeeey, success!'' |
| Line 312: | Line 312: | ||
'''5 September''' | '''5 September''' | ||
| − | - PCR on old colonies of LIP-RFP | + | - PCR on old colonies of LIP-RFP. |
| − | - Making LB-medium | + | - Making LB-medium. |
- Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies. | - Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies. | ||
| Line 320: | Line 320: | ||
'''6 September''' | '''6 September''' | ||
| − | - Cultivation of colony 8 (Gibson Assembly) with Hyg in the LB-media | + | - 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.'' | + | - Screening of colonies from Gibson Assembly. ''Observation: Bands were obtained, but no band was at 7000 bp.'' |
'''7 September''' | '''7 September''' | ||
| − | - PCR and gel electrophoresis on Hyg | + | - PCR and gel electrophoresis on Hyg. |
'''8 September''' | '''8 September''' | ||
| − | - Plasmid preparation of Gibson Assembly colony 8 | + | - Plasmid preparation of Gibson Assembly colony 8. |
| − | - Screening on Gibson colonies | + | - Screening on Gibson colonies. |
'''9 September''' | '''9 September''' | ||
| − | - '''''The sequences were obtained''''' ''We did not insert Hyg but instead YFP was inserted. Cas9 and LIP | + | - '''''The sequences were obtained''''' ''We did not insert Hyg but instead YFP was inserted. Cas9 and LIP eare inserted successfully!'' |
'''10 September''' | '''10 September''' | ||
| − | - Cultivation of algae mutants and Gibson colony 8 | + | - 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.'' | + | - Gel electrophoresis on plasmid preparation of Gibson colony 8 and Gibson colonies. ''Observation: The plasmid preparation of Gibson colony 8 showed good bands.'' |
'''11 September''' | '''11 September''' | ||
| − | - PCR on some Gibson colonies | + | - 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 | + | |
| Line 354: | Line 355: | ||
'''13 September''' | '''13 September''' | ||
| − | - OD measurments on the algae | + | - 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.'' | + | - Gel electrophoresis on the PCR product from 11/9 - 16. ''Observation: Band on 300 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp were obtained.'' |
'''14 September''' | '''14 September''' | ||
| − | - Dilution of the algae | + | - Dilution of the algae. |
| − | - Making TAP-40mM sucrose | + | - Making TAP-40mM sucrose. |
| − | - Plasmid preparation of Gibson Assembly colony 8 | + | - Plasmid preparation of Gibson Assembly colony 8. |
'''15 September''' | '''15 September''' | ||
| − | - Digestion of Gibson colony 8 | + | - Digestion of Gibson colony 8. |
'''16 September''' | '''16 September''' | ||
| − | - Electroporation on algae ''The algae have grown well.'' | + | - Electroporation on algae ''Observation: The algae have grown well.'' |
'''17 September''' | '''17 September''' | ||
| − | - PCR on Gibson colonies | + | - PCR on Gibson colonies. |
- Continuation on the electroporation from previous day. | - Continuation on the electroporation from previous day. | ||
| Line 382: | Line 383: | ||
'''18 September''' | '''18 September''' | ||
| − | - Gel electrophoresis on Gibson colonies | + | - Gel electrophoresis on Gibson colonies. |
| Line 388: | Line 389: | ||
'''19 September''' | '''19 September''' | ||
| − | - Sequenced was obtained. ''Looks like we did not insert U6 and sgRNA | + | - Sequenced was obtained. ''Observation: Looks like we did not insert U6 and sgRNA.'' |
'''21 September''' | '''21 September''' | ||
| − | - PCR on Gibson 3 | + | - PCR on Gibson 3. |
| − | - Gel electrophoresis on the PCR product from today. ''Band were obtained at 300 bp and 2000 bp.'' | + | - Gel electrophoresis on the PCR product from today. ''Observation: Band were obtained at 300 bp and 2000 bp.'' |
'''22 September''' | '''22 September''' | ||
| − | - Gel electrophoresis on PCR product from | + | - Gel electrophoresis on PCR product from previous day. ''Observation: Bands at 2000 bp and 300 bp were obtained'' |
- Gibson on pSB1C3 with LIP, LIP-RFP, U6 and UTR respectively. | - Gibson on pSB1C3 with LIP, LIP-RFP, U6 and UTR respectively. | ||
| − | - Transformation on all the Gibson product | + | - Transformation on all the Gibson product. |
'''23 September''' | '''23 September''' | ||
| − | - Gel electrophoresis on Gibson 3 colonies. ''No bands'' | + | - Gel electrophoresis on Gibson 3 colonies. ''Observation: No bands on the gel.'' |
- PCR of Gibson with LIP, LIP-RFP, U6 and Term. | - PCR of Gibson with LIP, LIP-RFP, U6 and Term. | ||
| − | - Screening of YFP transformed algae. ''No proof that the transformation worked'' | + | - Screening of YFP transformed algae. ''Observation: No proof that the transformation worked'' |
'''24 September''' | '''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 | + | - Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. ''Observation: Bands were obtained at 500 bp for Term, 600 bp for LIP and 1500 bp for LIP-RFP. No results for U6'' |
| − | - Gel electrophoresis on Gibson 3 colonies. ''No bands'' | + | - Gel electrophoresis on Gibson 3 colonies. ''Observation: No bands on the gel'' |
| − | - PCR on Gibson with U6, Term, LIP and LIP-RFP | + | - PCR on Gibson with U6, Term, LIP and LIP-RFP. |
| − | - Cultivation of U6, Term, LIP and LIP-RFP | + | - Cultivation of U6, Term, LIP and LIP-RFP. |
'''25 September''' | '''25 September''' | ||
| − | - PCR on Gibson 3 colonies | + | - PCR on Gibson 3 colonies. |
| − | - Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. ''Bands were obatined'' | + | - Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. ''Observation: Bands were obatined'' |
| − | - Cultivation of U6, Term, LIP and LIP-RFP | + | - Cultivation of U6, Term, LIP and LIP-RFP. |
| Line 434: | Line 435: | ||
'''26 September''' | '''26 September''' | ||
| − | - PCR on U6 colonies | + | - PCR on U6 colonies. |
| − | - Gel electrophoresis on Gibson 3 colonies. ''No | + | - Gel electrophoresis on Gibson 3 colonies. ''Observation: No results'' |
- Plasmid preparation on LIP, LIP-RFP and Term. | - Plasmid preparation on LIP, LIP-RFP and Term. | ||
| Line 444: | Line 445: | ||
- Plasmid preparation nr 2 on LIP, LIP-RFP and Term. | - Plasmid preparation nr 2 on LIP, LIP-RFP and Term. | ||
| − | - Gel electrophoresis on U6 colonies. ''No | + | - Gel electrophoresis on U6 colonies. ''Observation: No results'' |
| − | - PCR on Gibson 3 colonies | + | - PCR on Gibson 3 colonies. |
'''28 September''' | '''28 September''' | ||
| − | - Gel electrophoresis on Gibson 3 colonies. ''No result'' | + | - Gel electrophoresis on Gibson 3 colonies. ''Observation: No result'' |
| − | - Cultivation of U6, Term, LIP and LIP-RFP | + | - Cultivation of U6, Term, LIP and LIP-RFP. |
- PCR on Gibson 3 colonies. | - PCR on Gibson 3 colonies. | ||
| Line 458: | Line 459: | ||
'''29 September''' | '''29 September''' | ||
| − | - Gel electrophoresis | + | - Gel electrophoresis. |
'''1 October''' | '''1 October''' | ||
| − | - Gel electrophoresis on LIP, LIP-RFP and Terminator. ''Bands were obtained'' | + | - Gel electrophoresis on LIP, LIP-RFP and Terminator. ''Observation: Bands were obtained'' |
| − | - Plasmid preparation on LIP, LIP-RFP and Terminator | + | - Plasmid preparation on LIP, LIP-RFP and Terminator. |
| − | - New cultivation of LIP on plates | + | - New cultivation of LIP on plates. |
'''2 October''' | '''2 October''' | ||
| − | - Gel electrophoresis on LIP, LIP-RFP and Terminator. ''Bands were obtained'' | + | - Gel electrophoresis on LIP, LIP-RFP and Terminator. ''Observation: Bands were obtained'' |
| Line 476: | Line 477: | ||
'''3 October''' | '''3 October''' | ||
| − | - Sequencing of LIP, LIP-RFP and Term | + | - Sequencing of LIP, LIP-RFP and Term. |
'''5 October''' | '''5 October''' | ||
| − | - Gibson Assembly on U6 | + | - Gibson Assembly on U6. |
| − | - Transformation on U6 | + | - Transformation on U6. |
'''6 October''' | '''6 October''' | ||
| − | - PCR on U6 | + | - PCR on U6. |
| − | - Cultivation of LIP-RFP | + | - Cultivation of LIP-RFP. |
'''7 October''' | '''7 October''' | ||
| − | - Gel electrophoresis on U6. ''No insert'' | + | - Gel electrophoresis on U6. ''Observation: No insert'' |
| − | - Cultivation of algae | + | - Cultivation of algae. |
| − | - Cultivation of LIP-RFP | + | - Cultivation of LIP-RFP. |
'''8 October''' | '''8 October''' | ||
| − | - Cultivation of LIP-RFP | + | - Cultivation of LIP-RFP. |
'''9 October''' | '''9 October''' | ||
| − | - Plasmid preparation on LIP-RFP | + | - Plasmid preparation on LIP-RFP. |
{{Linkoping_Sweden/Footer}} | {{Linkoping_Sweden/Footer}} | ||
Revision as of 07:56, 16 October 2016
Contents
Overview on Laboration
Week 1
14 June
- First day at the lab! Making Hutner’s trace elements.
Week 2
21 June
- Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates.
Week 3
27 June
- Transformation of E1010. Observation: The transformation was successful.
28 June
- Control of competent cells.
29 June
- Transformation of E1010 to super competent XL-1. Observation: 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 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. Observation: 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 July
- PCR on Cas9.
12 July
- Gel electrophoresis on Cas9 to see if the PCR was successful. 'Observation: No bands were obtained for Cas9.
13 July
- PCR
14 July
- PCR on pSB1C3.
15 July
- Gel electrophoresis on pSB1C3. Observation: No bands were obtained on the gel.
- PCR on pSB1C3.
Week 6
18 July
- PCR and gel electrophoresis on pSB1C3. Observation: No bands were obtained on the gel.
20 July
- PCR and gel electrophoresis on pSB1C3. Observation: Bands were obtained 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 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. Observation: No bands were obtained on the gel.
- Digestion and Ligation on LIP-RFP and pSB1C3.
27 July
- New project approach
- Transformation of LIP-RFP and pSB1C3.
Week 8
1 August
- Cultivation of Hygromycin.
- Gel electrophoresis on UTR and LIP. Observation: Bands were obtained on the gel at 700 bp.
3 August
- Plasmid preparation of LIP, UTR and Hyg. Observation: Turned out to be incorrect later on.
- Transformation of LIP-RFP, U6, sgRNA and Cas9. Observation: 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 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. Observation: No bands on the gel.
10 August
- PCR on LIP-RFP and sgRNA
- Gel electrophoresis on LIP-RFP. Observation: 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. Observation: Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp.
12 August
- Gel electrophoresis on Cas9 and U6. Observation: Cas9 showed no bands. U6 showed bands at 350 bp. U6 with primers should be at 515 bp.
Week 10
15 August
- Preparation of TAP agar. Observation: Because of difficulties with the gas no plates could be performed today.
- Gel electrophoresis on UTR, LIP, Hyg and pSB1C3. pSB1C3 showed a band 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. Observation: The gel showed a weak band on Cas9 around 4000 bp.
17 August
- Plasmid preparation on LIP-RFP, U6 and sgRNA. 'Observation: Turned out to be incorrect later on.
18 August
- Cultivation of algae for transformation. 'Observation: It took 5 days for the algae wild type to reach OD = 1,757. The mutant algae evaporated.
- Making TAP agar plates.
- Making TAP Hyg. plates.
Week 11
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. Observation: Bands for Cas9 and Hyg were obtained.
- PCR purification of Cas9.
25 August
- Gel electrophoresis on pSB1C3. Observation: Bands were detected at 2000 bp which corresponds to the size of pSB1C3
- PCR purification on pSB1C3.
- First Gibson Assembly!
- Transformation of Gibson Assembly. Observation: Colonies were obtained!
- PCR on Cas9, Hyg, pSB1C3.
26 August
- PCR on Gibson Assembly product and colonies from Gibson Assembly transformation.
- Chloramphenicol plates.
- Gel electrophoresis on PCR product from Gibson Assembly, Cas9, Hyg and pSB1C3. Observation: Bands were detected for all the DNAs!
Week 12
29 August
- Gel electrophoresis on Gibson Assembly colonies. Observation: 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 colonies on new plates.
- Ligation on LIP-RFP with pSB1C3.
- New Gibson Assembly transformation.
31 August
- Gel electrophoresis on Gibson Assembly colonies. Observation: No bands on the gel.
- 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. Observation: No bands on the gel.
2 September
- Second Gibson assembly.
- Gibson transformation.
- Transformation of LIP-RFP.
3 September
- PCR and gel electrophoresis on gibson colonies. Observation: No results
- Digestion and ligation of LIP, U6, UTR, sgRNA and pSB1C3.
4 September
- PCR and gel electrophoresis on gibson colonies. Observation: It looks like Colony 8 has a band at 7000 bp! Yeeey, success!
Week 13
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. Observation: 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 eare inserted successfully!
10 September
- Cultivation of algae mutants and Gibson colony 8.
- Gel electrophoresis on plasmid preparation of Gibson colony 8 and Gibson colonies. Observation: 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
13 September
- OD measurments on the algae.
- Gel electrophoresis on the PCR product from 11/9 - 16. Observation: 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 Observation: 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 September
- Sequenced was obtained. Observation: Looks like we did not insert U6 and sgRNA.
21 September
- PCR on Gibson 3.
- Gel electrophoresis on the PCR product from today. Observation: Band were obtained at 300 bp and 2000 bp.
22 September
- Gel electrophoresis on PCR product from previous day. Observation: 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. Observation: No bands on the gel.
- PCR of Gibson with LIP, LIP-RFP, U6 and Term.
- Screening of YFP transformed algae. Observation: No proof that the transformation worked
24 September
- Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. Observation: Bands were obtained at 500 bp for Term, 600 bp for LIP and 1500 bp for LIP-RFP. No results for U6
- Gel electrophoresis on Gibson 3 colonies. Observation: No bands on the gel
- 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. Observation: Bands were obatined
- Cultivation of U6, Term, LIP and LIP-RFP.
Week 16
26 September
- PCR on U6 colonies.
- Gel electrophoresis on Gibson 3 colonies. Observation: No results
- 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. Observation: No results
- PCR on Gibson 3 colonies.
28 September
- Gel electrophoresis on Gibson 3 colonies. Observation: 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. Observation: 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. Observation: Bands were obtained
Week 17
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. Observation: No insert
- Cultivation of algae.
- Cultivation of LIP-RFP.
8 October
- Cultivation of LIP-RFP.
9 October
- Plasmid preparation on LIP-RFP.
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