Chlamydomonas can be transformed a number of ways. We generally use two methods in this laboratory, as seen below. Also, bacterial transformations are given.Chlamydomonas can be transformed a number of ways. We generally use two methods in this laboratory, as seen below. Also, bacterial transformations are given.Emetine/Gamete Step Transformation
Julie A.E. Nelson 3/23/93
1) Grow cells to good density in bubbler with 250ml SGII; count cells.
2) Pellet cells in IEC, resuspend in 1.5ml SGII/NO3.
3) Transfer cells to 50ml flask, measuring the new volume.
4) Shake under good light for 4 hours.
5) Dilute cells to 1.7*108 cells/ml and aliquot 0.3ml cells to 15ml conical tubes containing 0.3g sterile glass beads.
6) Add 2m g linearized DNA. Let the tubes sit at least 5 minutes.
7) Add 100m l 20% PEG and vortex at full speed for 45 seconds.
8) Add 10ml SGII/NO3, invert and pour cells into new tube.
9) Pellet cells in clinical centrifuge, pour off media.
10) Resuspend in 10ml SGII.
11) Lay tubes on sides and shake slowly under light for 3.5 hours.
12) Pellet cells and resuspend in 10ml SGII-N.
13) Pellet cells again and resuspend in 0.5ml SGII-N.
14) Transfer cells to 250ml bubbler with 100ml SGII-N
15) Bubble 1-4 days.
16) Pellet cells in IEC, resuspend in 40ml SGII.
17) Transfer cells to 250ml flask.
18) Shake under light for 8 hours.
19) Pellet cells, resuspend in 0.5ml SGII, plate on 100m g/ml emetine R plate.
Transformation of pET vectors into BL21 (DE3)/BL21 (DE3) plys competent Cells
1. Thaw vial of cells on ice.
2. Add 5 ml of DNA (pET vector + insert construct) directly to cells and stir gently with pipette tip to mix.
3. Incubate cells on ice for 5 min.
4. Heat shock the cells for exactly 30 seconds in a 42 oC water bath.
5. Return the cells to ice for 2 min.
6. Add 80 ml of room temperature SOC medium.
7. Shake cells for 1 hour at 37 oC.
8. Plate out 40 ml of cells on selective media (LB agar + 30 mg/ml kanamycin and 40 ml X-gal)
9. Grow overnight at 37 oC.
10. Select single white colonies for analysis of presence and orientation of insert.
Transformation of pCR vector products in to One Shot cells
1. Thaw vial(s) of One Shot cells on ice.
2. Add 2 ml of 0.5 M b-ME to cells and mix by gently stirring with pipette tip.
3. Add 5 ml of ligation reaction into cells and stir gently.
4. Incubate cells on ice for 30 min.
5. Heat shock cells by immersing in 42 oC water bath for exactly 30 seconds.
6. Return cells to ice for 2 min.
7. Add 250 ml of Invitrogen SOC medium.
8. Shake vial of cells in 37 oC shaker for 1 hour then return cells to ice.
Analysis
1. Plate 40 ml of transformed cells on an LB plate containing 50 mg/ml ampicillin and X-gal.
2. Incubate at 37 oC overnight.
3. Shift plate to 4 oC for color development.
4. Select single white colonies for analysis for the presence and orientation of insert by restriction mapping or sequencing.