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Line 17: | Line 17: | ||
'''21 June''' | '''21 June''' | ||
− | - Making SOC | + | - Making SOC medium, LB medium, LB agar and chloramphenicol plates. |
Line 23: | Line 23: | ||
'''27 June''' | '''27 June''' | ||
− | - Transformation of [http://parts.igem.org/Part:BBa_E1010 BBa_E1010] to test | + | - Transformation of [http://parts.igem.org/Part:BBa_E1010 BBa_E1010] to test transformation protocol. ''Observation: The transformation was successful.'' |
'''28 June''' | '''28 June''' | ||
Line 35: | Line 35: | ||
'''30 June''' | '''30 June''' | ||
− | - Making new E.Coli Calcium | + | - Making new ''E.Coli'' Calcium chloride competent cells. |
'''1 July''' | '''1 July''' | ||
− | - Making solutions for TAP | + | - Making solutions for TAP and TRIS medium. |
− | - Cultivation of | + | - Cultivation of successful transformants. |
Line 47: | Line 47: | ||
'''4 July''' | '''4 July''' | ||
− | - Making LB | + | - Making LB medium and LB agar. |
− | - Plasmid preparation of | + | - Plasmid preparation of succeded transformants week 3. |
- Test cultivation of algae. | - Test cultivation of algae. | ||
Line 57: | Line 57: | ||
- Making agar plates. | - Making agar plates. | ||
− | - | + | - Starting standard assembly of [https://2016.igem.org/Team:Linkoping_Sweden/Design construct]; digestion and ligation of pLIP ([http://parts.igem.org/Part:BBa_K2095000 BBa_K2095000]), U6 promoter, RBCS2 terminator ([http://parts.igem.org/Part:BBa_K2095002 K2095002]) and LIP-RFP ([http://parts.igem.org/Part:BBa_K2095003 BBa_K2095003]). |
− | - | + | - Competent cell test. |
- First algae cultivation. | - First algae cultivation. | ||
Line 65: | Line 65: | ||
'''6 July''' | '''6 July''' | ||
− | - Transformation on U6, | + | - Transformation on U6, pLIP, LIP-RFP and RBCS2 terminator. ''Observation: Colonies for U6, pLIP and LIP-RFP were detected.'' |
'''7 July''' | '''7 July''' | ||
− | - Cultivation of U6, | + | - Cultivation of U6, pLIP, LIP-RFP colonies in LB medium with chloramphenicol. ''Observation: Transformation of insert did not succeed'' |
− | |||
− | |||
Line 84: | Line 82: | ||
- Gel electrophoresis on Cas9 to see if the PCR was successful. ''Observation: No bands were obtained for Cas9.'' | - Gel electrophoresis on Cas9 to see if the PCR was successful. ''Observation: No bands were obtained for Cas9.'' | ||
− | |||
− | |||
− | |||
− | |||
'''14 July''' | '''14 July''' | ||
− | - PCR on pSB1C3. | + | - PCR on pSB1C3 for more product. |
'''15 July''' | '''15 July''' | ||
Line 97: | Line 91: | ||
- Gel electrophoresis on pSB1C3. ''Observation: No bands were obtained on the gel.'' | - Gel electrophoresis on pSB1C3. ''Observation: No bands were obtained on the gel.'' | ||
− | |||
Line 107: | Line 100: | ||
'''20 July''' | '''20 July''' | ||
− | - PCR and gel electrophoresis on pSB1C3. ''Observation: | + | - PCR and gel electrophoresis on pSB1C3. ''Observation: Correct bands were obtained - PCR on pSB1C3 succeeded.'' |
'''22 July''' | '''22 July''' | ||
− | - Digestion | + | - Digestion, ligation and transformation on pLIP, U6, RBCS2 terminator, Cas9, LIP-RFP, sgRNA and pSB1C3. |
Line 117: | Line 110: | ||
'''25 July''' | '''25 July''' | ||
− | - PCR | + | - PCR and recultivation of transformed pLIP and RBCS2 terminator colonies. |
− | + | ||
− | + | ||
− | - PCR purification. | + | - PCR purification of pSB1C3. |
'''26 July''' | '''26 July''' | ||
− | - Gel electrophoresis on pSB1C3, | + | - Gel electrophoresis on pSB1C3, pLIP, RBCS2. ''Observation: No bands were obtained on the gel.'' |
− | - Digestion and | + | - Digestion and ligation on LIP-RFP and pSB1C3. |
'''27 July''' | '''27 July''' | ||
− | - '''''New project approach''''' | + | - '''''New project approach''''' - Left standard assembly for Gibson assembly |
- Transformation of LIP-RFP and pSB1C3. | - Transformation of LIP-RFP and pSB1C3. | ||
Line 140: | Line 131: | ||
'''1 August''' | '''1 August''' | ||
− | - Cultivation of | + | - Cultivation of hygromycin-containing bacteria in order to isolate hygromycin which would be utilized to test electroporation in ''C.Reinhardtii'' |
− | - Gel electrophoresis on | + | - Gel electrophoresis on pLIP and RBCS2 terminator. ''Observation: Bands were obtained on the gel at 700 bp, indicates insert.'' |
'''3 August''' | '''3 August''' | ||
− | - Plasmid preparation of | + | - Plasmid preparation of pLIP, RBCS2 terminator and hygromycin. ''Observation: Turned out to be incorrect later on.'' |
− | - Transformation of LIP-RFP, U6, sgRNA and Cas9. ''Observation: 4 colonies for sgRNA and 4 colonies for LIP-RFP | + | - Transformation of LIP-RFP, U6, sgRNA and Cas9. ''Observation: 4 colonies for sgRNA and 4 colonies for LIP-RFP were obtained, but unfortunately no colonies for Cas9 and U6.'' |
'''4 August''' | '''4 August''' | ||
Line 159: | Line 150: | ||
'''8 August''' | '''8 August''' | ||
− | - PCR on | + | - PCR and recultivation on transformed LIP-RFP and sgRNA colonies. |
− | + | ||
− | + | ||
- First algae cultivation in darkness. | - First algae cultivation in darkness. | ||
Line 167: | Line 156: | ||
'''9 August''' | '''9 August''' | ||
− | - Transformation of | + | - Transformation of RBCS2 terminator and Cas9. |
− | - Gel electrophoresis on LIP-RFP and sgRNA. ''Observation: No bands | + | - Gel electrophoresis on LIP-RFP and sgRNA. ''Observation: No bands were obtained.'' |
'''10 August''' | '''10 August''' | ||
− | - PCR on LIP-RFP and sgRNA | + | - 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.'' | - 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.'' | ||
Line 181: | Line 170: | ||
- PCR on U6 and Cas9. | - PCR on U6 and Cas9. | ||
− | - Gel electrophoresis on sgRNA. ''Observation: | + | - Gel electrophoresis on sgRNA. ''Observation: No correct bands were obtained.'' |
'''12 August''' | '''12 August''' | ||
− | - Gel electrophoresis on Cas9 and U6. ''Observation: | + | - Gel electrophoresis on Cas9 and U6. ''Observation: No correct bands were obtained.'' |
Line 193: | Line 182: | ||
- Preparation of TAP agar. ''Observation: 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 | + | - Gel electrophoresis on RBCS2 terminator, pLIP, hygromycin and pSB1C3. ''Observation: No correct bands were obtained except for pSB1C3.'' |
− | - | + | - New cultivation of hygromycin-containing bacteria. |
'''16 August''' | '''16 August''' | ||
− | - Cultivation of sgRNA, LIP-RFP and U6. | + | - Cultivation of sgRNA, LIP-RFP and U6 transformants. |
− | - PCR on Cas9 and | + | - PCR on Cas9 and hygromycin. |
− | - Gel electrophoresis on Cas9 and | + | - Gel electrophoresis on Cas9 and hygromycin. ''Observation: The gel showed a weak band for Cas9 around 4000 bp.'' |
'''17 August''' | '''17 August''' | ||
− | - Plasmid preparation | + | - Plasmid preparation of LIP-RFP, U6 and sgRNA. ''Observation: Turned out to be incorrect later on.'' |
'''18 August''' | '''18 August''' | ||
− | - Cultivation of algae for transformation. ''Observation: It took 5 days for the | + | - Cultivation of algae for transformation. ''Observation: It took 5 days for the wild type algae to reach OD = 1,757. The mutant algae evaporated.'' |
- Making TAP agar plates. | - Making TAP agar plates. | ||
Line 222: | Line 211: | ||
'''22 August''' | '''22 August''' | ||
− | - Cultivation of LIP-RFP and | + | - Cultivation of LIP-RFP and hygromycin. |
'''23 August''' | '''23 August''' | ||
− | - Plasmid preparation | + | - Plasmid preparation of LIP-RFP and hygromycin-containing bacteria. |
- PCR on Cas9 and pSB1C3. | - PCR on Cas9 and pSB1C3. | ||
Line 234: | Line 223: | ||
'''24 August''' | '''24 August''' | ||
− | - PCR on LIP-RFP, | + | - PCR on LIP-RFP, hygromycin and pSB1C3. |
− | - Gel electrophoresis on Cas9, | + | - Gel electrophoresis on Cas9, hygromycin, LIP-RFP and pSB1C3. ''Observation: Bands for Cas9 and hygromycin were obtained.'' |
- PCR purification of Cas9. | - PCR purification of Cas9. | ||
Line 244: | Line 233: | ||
- Gel electrophoresis on pSB1C3. ''Observation: Bands were detected at 2000 bp which corresponds to the size of pSB1C3.'' | - Gel electrophoresis on pSB1C3. ''Observation: Bands were detected at 2000 bp which corresponds to the size of pSB1C3.'' | ||
− | - PCR purification | + | - PCR purification of pSB1C3. |
- '''''First Gibson Assembly!''''' | - '''''First Gibson Assembly!''''' | ||
Line 250: | Line 239: | ||
- Transformation of Gibson Assembly. ''Observation: Colonies were obtained!'' | - Transformation of Gibson Assembly. ''Observation: Colonies were obtained!'' | ||
− | - PCR on Cas9, | + | - PCR on Cas9, hygromycin, pSB1C3. |
'''26 August''' | '''26 August''' | ||
− | - PCR | + | - PCR and recultivation of Gibson Assembly transformants. |
- Chloramphenicol plates. | - Chloramphenicol plates. | ||
− | - Gel electrophoresis on PCR product from Gibson Assembly, Cas9, | + | - Gel electrophoresis on PCR product from Gibson Assembly, Cas9, hygromycin and pSB1C3. ''Observation: Bands were detected for all the DNAs!'' |
Line 264: | Line 253: | ||
'''29 August''' | '''29 August''' | ||
− | - Gel electrophoresis on Gibson Assembly colonies. ''Observation: Band at 5500 bp was obtained | + | - Gel electrophoresis on Gibson Assembly colonies. ''Observation: Band at 5500 bp was obtained, 7000 bp would indicate complete insert.'' |
− | - | + | - A new digestion on LIP-RFP. |
− | - PCR on Gibson Assembly colonies. | + | - PCR screening on Gibson Assembly colonies. |
'''30 August''' | '''30 August''' | ||
− | - PCR on Gibson Assembly colonies. | + | - PCR screening on Gibson Assembly colonies. |
- Cultivation of Gibson Assembly colonies on new plates. | - Cultivation of Gibson Assembly colonies on new plates. | ||
− | - Ligation on LIP-RFP with pSB1C3. | + | - Ligation and transformation on LIP-RFP with pSB1C3. |
- New Gibson Assembly transformation. | - New Gibson Assembly transformation. | ||
Line 282: | Line 271: | ||
'''31 August''' | '''31 August''' | ||
− | - Gel electrophoresis on Gibson Assembly colonies. ''Observation: | + | - Gel electrophoresis on Gibson Assembly colonies. ''Observation: The bands indicate that there is no insert.'' |
− | - Plasmid preparation | + | - Plasmid preparation of Gibson Assembly colony (2016-08-29). |
'''1 September''' | '''1 September''' | ||
− | - PCR on plasmid prepared U6, | + | - PCR on plasmid prepared U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin. |
− | - Gel electrophoresis on | + | - Gel electrophoresis on U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin ''Observation: No bands were obtained.'' |
'''2 September''' | '''2 September''' | ||
− | - Second Gibson | + | - Second Gibson Assembly. |
- Gibson transformation. | - Gibson transformation. | ||
Line 302: | Line 291: | ||
'''3 September''' | '''3 September''' | ||
− | - PCR and gel electrophoresis on Gibson colonies. ''Observation: No | + | - PCR and gel electrophoresis on Gibson colonies. ''Observation: No bands were obtained on the gel.'' |
− | - Digestion and ligation of | + | - Digestion and ligation of pLIP, U6, RBCS2 terminator, sgRNA and pSB1C3. |
'''4 September''' | '''4 September''' | ||
− | - PCR and gel electrophoresis on Gibson colonies. ''Observation: | + | - PCR and gel electrophoresis on Gibson colonies. ''Observation: Band from one colony (colony 8) indicates correct insert!'' |
Line 316: | Line 305: | ||
- PCR on old colonies of LIP-RFP. | - PCR on old colonies of LIP-RFP. | ||
− | - Making LB | + | - Making LB medium. |
- Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies. | - Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies. | ||
Line 322: | Line 311: | ||
'''6 September''' | '''6 September''' | ||
− | - Cultivation of colony 8 (Gibson Assembly) with | + | - Cultivation of colony 8 (Gibson Assembly) with hygromycin in the LB media. |
- Screening of colonies from Gibson Assembly. ''Observation: 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.'' | ||
Line 328: | Line 317: | ||
'''7 September''' | '''7 September''' | ||
− | - PCR and gel electrophoresis on | + | - PCR and gel electrophoresis on hygromycin. |
'''8 September''' | '''8 September''' | ||
Line 337: | Line 326: | ||
'''9 September''' | '''9 September''' | ||
− | + | ||
+ | '''''The sequence from colony 8 was obtained.''''' ''We did not insert hygromycin but instead YFP was inserted, this due to a 50% chance of amplifying either hygromycin or YFP when using PCR. Cas9 and pLIP were inserted successfully!'' | ||
'''10 September''' | '''10 September''' | ||
Line 343: | Line 333: | ||
- Cultivation of algae mutants and Gibson colony 8. | - Cultivation of algae mutants and Gibson colony 8. | ||
− | - Gel electrophoresis on plasmid | + | - Gel electrophoresis on Gibson colonies and plasmid prepared colony 8. ''Observation: The plasmid preparation of Gibson colony 8 showed good bands.'' |
'''11 September''' | '''11 September''' | ||
− | - PCR on | + | - PCR on Gibson colonies. |
− | - | + | - Recultivation of Gibson Assembly colonies on new plates. |
− | + | ||
− | + | ||
Line 359: | Line 347: | ||
- OD measurements on the algae. | - OD measurements on the algae. | ||
− | - Gel electrophoresis on the PCR product from 11/9 - 16. ''Observation: | + | - Gel electrophoresis on the PCR product from 11/9 - 16. ''Observation: No correct bands were obtained.'' |
'''14 September''' | '''14 September''' | ||
Line 371: | Line 359: | ||
'''15 September''' | '''15 September''' | ||
− | - Digestion of Gibson colony 8. | + | - Digestion of Gibson colony 8, for the electroporation. |
'''16 September''' | '''16 September''' | ||
− | - Electroporation on algae ''Observation: The algae have grown well.'' | + | - Electroporation on algae with Gibson colony 8 as the vector ''Observation: The algae have grown well.'' |
'''17 September''' | '''17 September''' | ||
− | - PCR on Gibson colonies. | + | - PCR screening on Gibson colonies. |
- Continuation on the electroporation from previous day. | - Continuation on the electroporation from previous day. | ||
Line 385: | Line 373: | ||
'''18 September''' | '''18 September''' | ||
− | - Gel electrophoresis on Gibson colonies. | + | - Gel electrophoresis on Gibson colonies from previous day. |
Line 391: | Line 379: | ||
'''19 September''' | '''19 September''' | ||
− | - | + | - The sequence from Gibson colony 8 was obtained. ''Observation: Looks like we did not insert U6 and sgRNA.'' |
+ | |||
+ | '''20 September''' | ||
+ | |||
+ | - Third Gibson Assembly. | ||
+ | |||
+ | - Gibson transformation. | ||
'''21 September''' | '''21 September''' | ||
− | - PCR on Gibson | + | - PCR screening on the third Gibson Assembly. |
- Gel electrophoresis on the PCR product from today. ''Observation: 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.'' | ||
Line 401: | Line 395: | ||
'''22 September''' | '''22 September''' | ||
− | - Gel electrophoresis on PCR product from previous day. ''Observation: | + | - Gel electrophoresis on PCR product from previous day. ''Observation: No correct bands were obtained.'' |
− | - Gibson on pSB1C3 with | + | - A new Gibson Assembly but only on pSB1C3 with pLIP, LIP-RFP, U6 and RBCS2 terminator respectively. |
− | - | + | - Gibson transformation. |
'''23 September''' | '''23 September''' | ||
− | - Gel electrophoresis on | + | - Gel electrophoresis on the obtained colonies from the third Gibson. ''Observation: No bands were detected on the gel.'' |
− | - PCR of Gibson with | + | - PCR of Gibson with pLIP, LIP-RFP, U6 and RBCS2 terminator. |
- Screening of YFP transformed algae. ''Observation: No proof that the transformation worked.'' | - Screening of YFP transformed algae. ''Observation: No proof that the transformation worked.'' | ||
Line 417: | Line 411: | ||
'''24 September''' | '''24 September''' | ||
− | - Gel electrophoresis on Gibson with U6, | + | - Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. ''Observation: Bands were obtained at 500 bp for RBCS2 terminator, 600 bp for pLIP and 1500 bp for LIP-RFP. No result for U6.'' |
− | - Gel electrophoresis on | + | - Gel electrophoresis on the colonies obtained from the third Gibson. ''Observation: The bands indicate that there was no insert.'' |
− | - PCR on Gibson with U6, | + | - PCR on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. |
− | - Cultivation of U6, | + | - Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP. |
'''25 September''' | '''25 September''' | ||
− | - PCR on | + | - PCR on the colonies obtained from the third Gibson. |
− | - Gel electrophoresis on Gibson with U6, | + | - Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. ''Observation: Bands were obtained for all the DNA fragments except from U6'' |
− | - Cultivation of U6, | + | - Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP. |
Line 439: | Line 433: | ||
- PCR on U6 colonies. | - PCR on U6 colonies. | ||
− | - Gel electrophoresis on | + | - Gel electrophoresis on the colonies obtained from the third Gibson. ''Observation: The bands indicate that there is no insert.'' |
− | - Plasmid preparation | + | - Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator. |
'''27 September''' | '''27 September''' | ||
− | - Plasmid preparation nr 2 | + | - Plasmid preparation nr 2 of pLIP, LIP-RFP and RBCS2 terminator. |
- Gel electrophoresis on U6 colonies. ''Observation: No results.'' | - Gel electrophoresis on U6 colonies. ''Observation: No results.'' | ||
− | - PCR on | + | - PCR on the colonies obtained from the third Gibson. |
'''28 September''' | '''28 September''' | ||
− | - Gel electrophoresis on | + | - Gel electrophoresis on the colonies obtained from the third Gibson. ''Observation: No results.'' |
− | - Cultivation of U6, | + | - Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP. |
− | - PCR on | + | - PCR on the colonies obtained from the third Gibson. |
'''29 September''' | '''29 September''' | ||
− | - Gel electrophoresis. | + | - Gel electrophoresis on the PCR product from the previous day. ''Observation: The bands indicate that there is no insert.'' |
'''1 October''' | '''1 October''' | ||
− | - Gel electrophoresis on | + | - Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. ''Observation: Correct bands were obtained!.'' |
− | - Plasmid preparation | + | - Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator. |
− | - New cultivation of | + | - New cultivation of pLIP on plates. |
'''2 October''' | '''2 October''' | ||
− | - Gel electrophoresis on | + | - Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. ''Observation: Correct bands were obtained!'' |
Line 479: | Line 473: | ||
'''3 October''' | '''3 October''' | ||
− | - Sequencing of | + | - Sequencing of pLIP, LIP-RFP and RBCS2 terminator. |
'''5 October''' | '''5 October''' | ||
− | - Gibson Assembly on U6. | + | - New Gibson Assembly on pSB1C3 with U6. |
− | - | + | - Gibson transformation on pSB1C3 with U6. |
'''6 October''' | '''6 October''' | ||
Line 495: | Line 489: | ||
'''7 October''' | '''7 October''' | ||
− | - Gel electrophoresis on U6. ''Observation: | + | - Gel electrophoresis on U6. ''Observation: The band indicate that there is no insert.'' |
- Cultivation of algae. | - Cultivation of algae. | ||
− | |||
− | |||
'''8 October''' | '''8 October''' | ||
Line 507: | Line 499: | ||
'''9 October''' | '''9 October''' | ||
− | - Plasmid preparation | + | - Plasmid preparation of LIP-RFP. |
Line 513: | Line 505: | ||
'''11 October''' | '''11 October''' | ||
− | - Plasmid preparation | + | - Plasmid preparation of pLIP and RBCS2 terminator. |
'''12 October''' | '''12 October''' | ||
− | - Gel electrophoresis on | + | - Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. '''''This was the last day at the lab!''''' |
+ | |||
+ | ===Week 19=== | ||
+ | '''19 October''' | ||
+ | |||
+ | - Parts were finally prepared for submission! | ||
+ | |||
+ | '''20 October''' | ||
+ | - Parts submitted | ||
+ | |||
{{Linkoping_Sweden/Footer}} | {{Linkoping_Sweden/Footer}} |
Latest revision as of 17:46, 19 October 2016
Contents
Experiments
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 [http://parts.igem.org/Part:BBa_E1010 BBa_E1010] to test transformation protocol. Observation: The transformation was successful.
28 June
- Control of competent cells.
29 June
- Transformation of BBa_E1010 to super competent XL-1. Observation: The transformation was successful.
30 June
- Making new E.Coli Calcium chloride competent cells.
1 July
- Making solutions for TAP and TRIS medium.
- Cultivation of successful transformants.
Week 4
4 July
- Making LB medium and LB agar.
- Plasmid preparation of succeded transformants week 3.
- Test cultivation of algae.
5 July
- Making agar plates.
- Starting standard assembly of construct; digestion and ligation of pLIP ([http://parts.igem.org/Part:BBa_K2095000 BBa_K2095000]), U6 promoter, RBCS2 terminator ([http://parts.igem.org/Part:BBa_K2095002 K2095002]) and LIP-RFP ([http://parts.igem.org/Part:BBa_K2095003 BBa_K2095003]).
- Competent cell test.
- First algae cultivation.
6 July
- Transformation on U6, pLIP, LIP-RFP and RBCS2 terminator. Observation: Colonies for U6, pLIP and LIP-RFP were detected.
7 July
- Cultivation of U6, pLIP, LIP-RFP colonies in LB medium with chloramphenicol. Observation: Transformation of insert did not succeed
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.
14 July
- PCR on pSB1C3 for more product.
15 July
- Gel electrophoresis on pSB1C3. Observation: No bands were obtained on the gel.
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: Correct bands were obtained - PCR on pSB1C3 succeeded.
22 July
- Digestion, ligation and transformation on pLIP, U6, RBCS2 terminator, Cas9, LIP-RFP, sgRNA and pSB1C3.
Week 7
25 July
- PCR and recultivation of transformed pLIP and RBCS2 terminator colonies.
- PCR purification of pSB1C3.
26 July
- Gel electrophoresis on pSB1C3, pLIP, RBCS2. Observation: No bands were obtained on the gel.
- Digestion and ligation on LIP-RFP and pSB1C3.
27 July
- New project approach - Left standard assembly for Gibson assembly
- Transformation of LIP-RFP and pSB1C3.
Week 8
1 August
- Cultivation of hygromycin-containing bacteria in order to isolate hygromycin which would be utilized to test electroporation in C.Reinhardtii
- Gel electrophoresis on pLIP and RBCS2 terminator. Observation: Bands were obtained on the gel at 700 bp, indicates insert.
3 August
- Plasmid preparation of pLIP, RBCS2 terminator and hygromycin. Observation: Turned out to be incorrect later on.
- Transformation of LIP-RFP, U6, sgRNA and Cas9. Observation: 4 colonies for sgRNA and 4 colonies for LIP-RFP were obtained, but unfortunately no colonies for Cas9 and U6.
4 August
- Making TAP medium for cultivation of algae in the dark.
Week 9
8 August
- PCR and recultivation on transformed LIP-RFP and sgRNA colonies.
- First algae cultivation in darkness.
9 August
- Transformation of RBCS2 terminator and Cas9.
- Gel electrophoresis on LIP-RFP and sgRNA. Observation: No bands were obtained.
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: No correct bands were obtained.
12 August
- Gel electrophoresis on Cas9 and U6. Observation: No correct bands were obtained.
Week 10
15 August
- Preparation of TAP agar. Observation: Because of difficulties with the gas no plates could be performed today.
- Gel electrophoresis on RBCS2 terminator, pLIP, hygromycin and pSB1C3. Observation: No correct bands were obtained except for pSB1C3.
- New cultivation of hygromycin-containing bacteria.
16 August
- Cultivation of sgRNA, LIP-RFP and U6 transformants.
- PCR on Cas9 and hygromycin.
- Gel electrophoresis on Cas9 and hygromycin. Observation: The gel showed a weak band for Cas9 around 4000 bp.
17 August
- Plasmid preparation of 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 wild type algae 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 hygromycin.
23 August
- Plasmid preparation of LIP-RFP and hygromycin-containing bacteria.
- PCR on Cas9 and pSB1C3.
- New cultivation of algae in the dark.
24 August
- PCR on LIP-RFP, hygromycin and pSB1C3.
- Gel electrophoresis on Cas9, hygromycin, LIP-RFP and pSB1C3. Observation: Bands for Cas9 and hygromycin 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 of pSB1C3.
- First Gibson Assembly!
- Transformation of Gibson Assembly. Observation: Colonies were obtained!
- PCR on Cas9, hygromycin, pSB1C3.
26 August
- PCR and recultivation of Gibson Assembly transformants.
- Chloramphenicol plates.
- Gel electrophoresis on PCR product from Gibson Assembly, Cas9, hygromycin and pSB1C3. Observation: Bands were detected for all the DNAs!
Week 12
29 August
- Gel electrophoresis on Gibson Assembly colonies. Observation: Band at 5500 bp was obtained, 7000 bp would indicate complete insert.
- A new digestion on LIP-RFP.
- PCR screening on Gibson Assembly colonies.
30 August
- PCR screening on Gibson Assembly colonies.
- Cultivation of Gibson Assembly colonies on new plates.
- Ligation and transformation on LIP-RFP with pSB1C3.
- New Gibson Assembly transformation.
31 August
- Gel electrophoresis on Gibson Assembly colonies. Observation: The bands indicate that there is no insert.
- Plasmid preparation of Gibson Assembly colony (2016-08-29).
1 September
- PCR on plasmid prepared U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin.
- Gel electrophoresis on U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin Observation: No bands were obtained.
2 September
- Second Gibson Assembly.
- Gibson transformation.
- Transformation of LIP-RFP.
3 September
- PCR and gel electrophoresis on Gibson colonies. Observation: No bands were obtained on the gel.
- Digestion and ligation of pLIP, U6, RBCS2 terminator, sgRNA and pSB1C3.
4 September
- PCR and gel electrophoresis on Gibson colonies. Observation: Band from one colony (colony 8) indicates correct insert!
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 hygromycin 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 hygromycin.
8 September
- Plasmid preparation of Gibson Assembly colony 8.
- Screening on Gibson colonies.
9 September
The sequence from colony 8 was obtained. We did not insert hygromycin but instead YFP was inserted, this due to a 50% chance of amplifying either hygromycin or YFP when using PCR. Cas9 and pLIP were inserted successfully!
10 September
- Cultivation of algae mutants and Gibson colony 8.
- Gel electrophoresis on Gibson colonies and plasmid prepared colony 8. Observation: The plasmid preparation of Gibson colony 8 showed good bands.
11 September
- PCR on Gibson colonies.
- Recultivation of Gibson Assembly colonies on new plates.
Week 14
13 September
- OD measurements on the algae.
- Gel electrophoresis on the PCR product from 11/9 - 16. Observation: No correct bands 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, for the electroporation.
16 September
- Electroporation on algae with Gibson colony 8 as the vector Observation: The algae have grown well.
17 September
- PCR screening on Gibson colonies.
- Continuation on the electroporation from previous day.
18 September
- Gel electrophoresis on Gibson colonies from previous day.
Week 15
19 September
- The sequence from Gibson colony 8 was obtained. Observation: Looks like we did not insert U6 and sgRNA.
20 September
- Third Gibson Assembly.
- Gibson transformation.
21 September
- PCR screening on the third Gibson Assembly.
- 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: No correct bands were obtained.
- A new Gibson Assembly but only on pSB1C3 with pLIP, LIP-RFP, U6 and RBCS2 terminator respectively.
- Gibson transformation.
23 September
- Gel electrophoresis on the obtained colonies from the third Gibson. Observation: No bands were detected on the gel.
- PCR of Gibson with pLIP, LIP-RFP, U6 and RBCS2 terminator.
- Screening of YFP transformed algae. Observation: No proof that the transformation worked.
24 September
- Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. Observation: Bands were obtained at 500 bp for RBCS2 terminator, 600 bp for pLIP and 1500 bp for LIP-RFP. No result for U6.
- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: The bands indicate that there was no insert.
- PCR on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP.
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
25 September
- PCR on the colonies obtained from the third Gibson.
- Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. Observation: Bands were obtained for all the DNA fragments except from U6
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
Week 16
26 September
- PCR on U6 colonies.
- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: The bands indicate that there is no insert.
- Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator.
27 September
- Plasmid preparation nr 2 of pLIP, LIP-RFP and RBCS2 terminator.
- Gel electrophoresis on U6 colonies. Observation: No results.
- PCR on the colonies obtained from the third Gibson.
28 September
- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: No results.
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
- PCR on the colonies obtained from the third Gibson.
29 September
- Gel electrophoresis on the PCR product from the previous day. Observation: The bands indicate that there is no insert.
1 October
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. Observation: Correct bands were obtained!.
- Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator.
- New cultivation of pLIP on plates.
2 October
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. Observation: Correct bands were obtained!
Week 17
3 October
- Sequencing of pLIP, LIP-RFP and RBCS2 terminator.
5 October
- New Gibson Assembly on pSB1C3 with U6.
- Gibson transformation on pSB1C3 with U6.
6 October
- PCR on U6.
- Cultivation of LIP-RFP.
7 October
- Gel electrophoresis on U6. Observation: The band indicate that there is no insert.
- Cultivation of algae.
8 October
- Cultivation of LIP-RFP.
9 October
- Plasmid preparation of LIP-RFP.
Week 18
11 October
- Plasmid preparation of pLIP and RBCS2 terminator.
12 October
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. This was the last day at the lab!
Week 19
19 October
- Parts were finally prepared for submission!
20 October - Parts submitted