Difference between revisions of "Team:Linkoping Sweden/Experiments"
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{{Linkoping_Sweden}} | {{Linkoping_Sweden}} | ||
| − | + | =Experiments - Overview on Laboration= | |
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==Week 1: 13 – 19 June== | ==Week 1: 13 – 19 June== | ||
| − | * 14 June: First day at the lab! Making Hutner’s trace elements | + | * '''14 June''': First day at the lab! Making Hutner’s trace elements |
==Week 2: 20 – 26 June== | ==Week 2: 20 – 26 June== | ||
| − | * 21 June: Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates. | + | * '''21 June''': Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates. |
- 46 agar plates were made. | - 46 agar plates were made. | ||
==Week 3: 27 June – 3 July== | ==Week 3: 27 June – 3 July== | ||
| − | * 27 June: Transformation of E1010 | + | * '''27 June''': Transformation of E1010 |
- The transformation was successful | - The transformation was successful | ||
| − | * 28 June: Control of competent cells | + | * '''28 June''': Control of competent cells |
| − | * 29 June: Transformation of E1010 to super competent XL-1 | + | * '''29 June''': Transformation of E1010 to super competent XL-1 |
- The transformation was successful. | - The transformation was successful. | ||
| − | * 30 June: Making E.Coli Calcium Chloride competent cells | + | * '''30 June''': 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 28: | Line 28: | ||
==Week 4: 4 – 10 July== | ==Week 4: 4 – 10 July== | ||
| − | * 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: Transformation on U6, LIP, LIP-RFP and UTR. | + | * '''6 July''': Transformation on U6, LIP, LIP-RFP and UTR. |
Colonies for U6 and LIP were detected. | Colonies for U6 and LIP were detected. | ||
| − | * 7 July: Cultivation of U6, LIP, LIP-RFP colonies on new plates with Chloramphenicol | + | * '''7 July''': Cultivation of U6, LIP, LIP-RFP colonies on new plates with Chloramphenicol |
| − | * 8 July: OD measurement of transformated bacteria. | + | * '''8 July''': OD measurement of transformated bacteria. |
==Week 5: 11 – 17 July== | ==Week 5: 11 – 17 July== | ||
| − | * 11 July: PCR on Cas9 | + | * '''11 July''': PCR on Cas9 |
| − | * 12 July: Gel electrophoresis on Cas9 to see if the PCR succeeded. | + | * '''12 July''': Gel electrophoresis on Cas9 to see if the PCR succeeded. |
The gel did not show any bands for Cas9 | The gel did not show any bands for Cas9 | ||
| − | * 13 July: PCR | + | * '''13 July''': PCR |
| − | * 14 July: PCR on pSB1C3 | + | * '''14 July''': PCR on pSB1C3 |
| − | * 15 July: | + | * '''15 July''': |
- Gel electrophoresis on pSB1C3 | - Gel electrophoresis on pSB1C3 | ||
No bands were obtained on the gel. | No bands were obtained on the gel. | ||
| Line 56: | Line 56: | ||
==Week 6: 18 – 24 July== | ==Week 6: 18 – 24 July== | ||
| − | * 18 July: PCR and gel electrophoresis on pSB1C3 | + | * '''18 July''': PCR and gel electrophoresis on pSB1C3 |
No bands were obtained. | No bands were obtained. | ||
| − | * 20 July: PCR and gel electrophoresis on pSB1C3 | + | * '''20 July''': PCR and gel electrophoresis on pSB1C3 |
We obtained bands on the gel at approximately 2000 bp. | 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. | + | * '''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: | + | * '''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 | - Gel electrophoresis on pSB1C3, UTR and LIP | ||
No bands were obtained. | No bands were obtained. | ||
- Digestion and Ligation on LIP-RFP and pSB1C3. | - Digestion and Ligation on LIP-RFP and pSB1C3. | ||
| − | * 27 July: New project approach | + | * '''27 July''': New project approach |
Transformation of LIP-RFP and pSB1C3. | Transformation of LIP-RFP and pSB1C3. | ||
==Week 8: 1 – 7 August== | ==Week 8: 1 – 7 August== | ||
| − | * 1 August: | + | * '''1 August''': |
- Cultivation of Hyg | - Cultivation of Hyg | ||
- Gel electrophoresis on UTR and LIP | - Gel electrophoresis on UTR and LIP | ||
Bands were obtained at 700 bp. | Bands were obtained at 700 bp. | ||
| − | * 3 August: | + | * '''3 August''': |
- Plasmid preparation of LIP, UTR and Hyg. | - Plasmid preparation of LIP, UTR and Hyg. | ||
Was later show to be wrong | Was later show to be wrong | ||
- Transformation of LIP-RFP, U6, sgRNA and Cas9. | - 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 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 | + | * '''4 August''': Making TAP medium for cultivation of algae in the dark |
==Week 9: 8 – 14 August== | ==Week 9: 8 – 14 August== | ||
| − | * 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''': |
- Transformation of U6 and Cas9. | - Transformation of U6 and Cas9. | ||
- Gel electrophoresis on LIP-RFP and sgRNA. | - Gel electrophoresis on LIP-RFP and sgRNA. | ||
No bands on the gel. | No bands on the gel. | ||
| − | * 10 August: | + | * '''10 August''': |
- PCR on LIP-RFP and sgRNA | - PCR on LIP-RFP and sgRNA | ||
- Gel electrophoresis on LIP-RFP | - 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. | Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good. | ||
| − | * 11 August: | + | * '''11 August''': |
- PCR on U6 and Cas9. | - PCR on U6 and Cas9. | ||
- Gel electrophoresis on sgRNA. | - Gel electrophoresis on sgRNA. | ||
Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp. | Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp. | ||
| − | * 12 August: Gel electrophoresis on Cas9 and U6. | + | * '''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. | 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: | + | * '''15 August''': |
- Preparation of TAP agar | - Preparation of TAP agar | ||
Because of difficulties with the gas no plates could be performed today. | Because of difficulties with the gas no plates could be performed today. | ||
| Line 117: | Line 117: | ||
pSB1C3 showed at 2000 bp as it was supposed to. The other fragments did not show any bands. | pSB1C3 showed at 2000 bp as it was supposed to. The other fragments did not show any bands. | ||
- Cultivation of Hyg. | - Cultivation of Hyg. | ||
| − | * 16 August: | + | * '''16 August''': |
- 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 | - Gel electrophoresis on Cas9 and Hyg | ||
The gel showed a weak band on Cas9 around 4000 bp. | The gel showed a weak band on Cas9 around 4000 bp. | ||
| − | * 17 August: Plasmid preparation on LIP-RFP, U6 and sgRNA | + | * '''17 August''': Plasmid preparation on LIP-RFP, U6 and sgRNA |
Was later show to be wrong | Was later show to be wrong | ||
| − | * 18 August: | + | * '''18 August''': |
- Cultivation of algae for transformation | - Cultivation of algae for transformation | ||
It took 5 days for the algae wild type to reach OD 1,757 | It took 5 days for the algae wild type to reach OD 1,757 | ||
| Line 133: | Line 133: | ||
==Week 11: 22 – 28 August== | ==Week 11: 22 – 28 August== | ||
| − | * 22 August: Cultivation of LIP-RFP and Hyg | + | * '''22 August''': 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. | - Gel electrophoresis on Cas9, Hyg, LIP-RFP and pSB1C3. | ||
Bands for Cas9 and Hyg were obtained. | Bands for Cas9 and Hyg were obtained. | ||
- PCR purification of Cas9. | - PCR purification of Cas9. | ||
| − | * 25 August: | + | * '''25 August''': |
- Gel electrophoresis on pSB1C3 | - Gel electrophoresis on pSB1C3 | ||
Bands were detected at 2000 bp which match with pSB1C3 | Bands were detected at 2000 bp which match with pSB1C3 | ||
| Line 151: | Line 151: | ||
Colonies were obtained! | 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 | ||
| Line 160: | Line 160: | ||
==Week 12: 29 August – 4 September== | ==Week 12: 29 August – 4 September== | ||
| − | * 29 August: | + | * '''29 August''': |
- Gel electrophoresis on Gibson Assembly colonies | - Gel electrophoresis on Gibson Assembly colonies | ||
A band at 5500 bp was obtained. We want bands at 7000 bp. | 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. | ||
| − | * 30 August: | + | * '''30 August''': |
- PCR on Gibson Assembly colonies. | - PCR on Gibson Assembly colonies. | ||
- Cultivation of Gibson Assembly coloni on new plates | - Cultivation of Gibson Assembly coloni 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 | - Gel electrophoresis on Gibson Assembly colonies | ||
No bands. | No bands. | ||
- Plasmid preparation on Gibson Assembly colony. | - Plasmid preparation on Gibson Assembly colony. | ||
| − | * 1 September: | + | * '''1 September''': |
- 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. | - Gel electrophoresis on LIP, sgRNA, U6, UTR, LIP-RFP and Hyg. | ||
No result on the gel. | No result on the gel. | ||
| − | * 2 September | + | * '''2 September''' |
- Second Gibson assembly | - Second Gibson assembly | ||
- Gibson transformation | - Gibson transformation | ||
- Transformation LIP-RFP | - Transformation LIP-RFP | ||
| − | * 3 September | + | * '''3 September''' |
- PCR and gel electrophoresis on gibson colonies | - PCR and gel electrophoresis on gibson colonies | ||
No results | 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 | - PCR and gel electrophoresis on gibson colonies | ||
It looks like Colony 8 has a band at 7000 bp! Yeeey! | It looks like Colony 8 has a band at 7000 bp! Yeeey! | ||
| Line 192: | Line 192: | ||
==Week 13: 5 – 11 September== | ==Week 13: 5 – 11 September== | ||
| − | * 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. | ||
| − | * 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 | Screening of colonies from Gibson Assembly | ||
Bands were obtained, but no band was at 7000 bp. | 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 | The sequences were obtained | ||
We did not insert Hyg but instead YFP was inserted. | We did not insert Hyg but instead YFP was inserted. | ||
Cas9 and LIP are inserted successfully! | Cas9 and LIP are 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 | Gel electrophoresis on plasmid preparation of Gibson colony 8 and Gibson colonies | ||
The plasmid preparation of Gibson colony 8 showed good bands. | 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 | Preparation for plasmid preparation | ||
| Line 221: | Line 221: | ||
==Week 14: 12 – 18 September== | ==Week 14: 12 – 18 September== | ||
| − | * 13 September | + | * '''13 September''' |
OD measurments on the algae | OD measurments on the algae | ||
Gel electrophoresis on the PCR product from 11/9 - 16. | Gel electrophoresis on the PCR product from 11/9 - 16. | ||
Band on 300 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp were obtained. | 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 | Electroporation on algae | ||
The algae have grown well. | 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. | ||
| − | * 18 September | + | * '''18 September''' |
Gel electrophoresis on Gibson colonies | Gel electrophoresis on Gibson colonies | ||
==Week 15: 19 – 25 September== | ==Week 15: 19 – 25 September== | ||
| − | * 19 September | + | * '''19 September''' |
Sequenced was obtained | Sequenced was obtained | ||
Looks like we did not insert U6 and sgRNA :( | 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 | Gel electrophoresis on the PCR product from today | ||
Band were obtained at 300 bp and 2000 bp. | Band were obtained at 300 bp and 2000 bp. | ||
| − | * 22 September | + | * '''22 September''' |
Gel electrophoresis on PCR product from yesterday | Gel electrophoresis on PCR product from yesterday | ||
Bands at 2000 bp and 300 bp were obtained | 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 | Gel electrophoresis on Gibson 3 colonies | ||
No bands. | No bands. | ||
| Line 260: | Line 260: | ||
Screening of YFP transformed algae | Screening of YFP transformed algae | ||
No proof that the transformation worked. | No proof that the transformation worked. | ||
| − | * 24 September | + | * '''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 obtained at 500 bp for Term, 600 bp for LIP and 1500 bp for LIP-RFP. No result for U6. | ||
| Line 267: | Line 267: | ||
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. | Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. | ||
| Line 275: | Line 275: | ||
==Week 16: 26 September - 2 October== | ==Week 16: 26 September - 2 October== | ||
| − | * 26 September | + | * '''26 September''' |
PCR on U6 colonies | PCR on U6 colonies | ||
Gel electrophoresis on Gibson 3 colonies | Gel electrophoresis on Gibson 3 colonies | ||
No result. | No result. | ||
Plasmid preparation on LIP, LIP-RFP and Term. | Plasmid preparation on LIP, LIP-RFP and Term. | ||
| − | * 27 September | + | * '''27 September''' |
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 | Gel electrophoresis on U6 colonies | ||
No result. | No result. | ||
PCR on Gibson 3 colonies | PCR on Gibson 3 colonies | ||
| − | * 28 September | + | * '''28 September''' |
Gel electrophoresis on Gibson 3 colonies | Gel electrophoresis on Gibson 3 colonies | ||
No result. | 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. | ||
| − | * 29 September | + | * '''29 September''' |
Gel electrophoresis | Gel electrophoresis | ||
| − | * 1 October | + | * '''1 October''' |
Gel electrophoresis on LIP, LIP-RFP and Terminator | Gel electrophoresis on LIP, LIP-RFP and Terminator | ||
Bands were obtained | 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 | Gel electrophoresis on LIP, LIP-RFP and Terminator | ||
Bands were obtained | Bands were obtained | ||
| Line 303: | Line 303: | ||
==Week 17: 3 - 9 October== | ==Week 17: 3 - 9 October== | ||
| − | * 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 | Gel electrophoresis on U6 | ||
No insert | 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 11:55, 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
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