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