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Revision as of 14:00, 13 October 2016
Contents
- 1 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
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