Cô lập protoplast trên Arabidopsis và rêu Physcomitrella

ARABIDOPSIS PROTOPLAST ISOLATION

Protoplast isolation steps:

-       Cut 0.5-1 mm leaf strip with fresh blades without wounding or use taping method to peel the lower epidermis of the leaves

-       Put leaves into petri disk containing 10 mL cellulose/macerozyme solution

-       Vacuum 5 min then incubate around 3h in darkness at 20⁰C for cutting method or around 1.5h for taping method

-       Add the same volume of W5 solution (~10mL) to petri dish

-       Filter protoplast suspension with a 35-75 um nylon mesh.

-       Spin at 100g for 2 min to pellet the protoplasts

-       Remove supernatant and resuspend protoplasts in W5 (5-10mL)

-       Count number of protoplast for dilution at 2x10⁵ protoplast/mL

-       Leave protoplasts settle down by themselves at RT, cover tubes by aluminum foil

-       Remove W5 solution and resuspend protoplast in MMg solution (3-6mL) at RT

-       Protoplast are ready for transformation

Transformation steps:

-       Add a certain DNA concentration to bottom of eppendorf

-       Add 100uL protoplast suspension and mix gently

-       Add 110uL PEG solution and mix gently

-       Incubate 15 min in darkness

-       Add 440uL W5 solution and mix gently

-       Centrifuge 100g at RT for 2 min

-       Remove supernatant

-       Add 1mL WI solution and mix gently

-       Transfer protoplast suspension to plate and incubate overnight at 20⁰C in darkness

-       Transfer protoplast suspension into eppendorf

-       Centrifuge 200g for 2 min

-       Remove supernatant, freeze and store samples at -80⁰C until for analysis

Dual luciferase assay reagents

PLB buffer 1X: dilute PLB buffer 5X by sterile water

Stop&Glo 1X: dilute 1 part substrate 50X + 49 part buffer

LAR II aliqot (store at -80⁰C)

Procedure:

-       Switch on laminator machine and choose the program: measure-protocol-number 2-enter

-       Add 30ul PLB 1X solution into protoplast tube (2mL) and mix well to produce lysate

-       Prepare glass tube (use for laminator) and add 30ul of LAR II at middle bottom without making bubble

-       Add 6ul lysate into glass tube containing LAR II, mix by pipetting and put into laminator for first reading. Wait until machine request for second reading

-       Take out glass tube and add 30ul Stop&Glo, mix by pipetting and put back again into laminator for second reading. This step should be done fast.

-       ……

-       Finish process and analyze your results.

Good luck!!!

Solutions for protoplast isolation

MOSS PROTOPLAST ISOLATION

1.    Collect protonema from 7 day culture and transfer to a 9cm pertri dish containing 10mL mannitol 8.5% (0.48M).

2.    Add 10mL of Drisilase 2% solution and incubate at room temperature for 30-60 min with occasional gentle mixing.

3.    Filter through a mesh into a 10mL tube or small beaker.

4.    Harvest protoplast by centrifuge at low speed 200g for 4 min.

5.    Gently and resuspend pellet in 10mL mannitol 8.5% and repeat centrifuge.

6.    Resuspend pellet in 10mL mannitol 8.5% and count. Leave the protoplasts in mannitol before initiating transformation.

7.    Recentrifuge 200g for 5 min and then resuspend the protoplasts at a conc of 1.2x10⁶/mL in MMM solution (conc 1 to 1.5x10⁶/mL are optimal for transformation efficiency).

A.  Transient assay

8.    Prepare 5µg of DNA into 2mL eppendorf.

9.    Add 100µL of protoplast suspension and 100 µL PEG solution. Mix by tapping the tube gently.

10. Heat the mixture at 45⁰C for 5 min.

11. Transfer the tube to room temp and leave for 5 min with occasional gentle mixing.

12. Add 100µL moss liquid medium. Gently invert the tube to mix. Wait at least 1 min

13. Repeat step 12 four times.

14. Add 300µL moss liquid medium. Invert gently to mix. Wait at least 1 min.

15. Repeat step 14 four times.

16. Keep the transformed protoplast overnight in darkness.

17. Harvest protoplasts for transient assay analysis.

B.  Regeneration of moss protoplast for stable transformation

Protoplasts in MMM solution is ready for the transformation

1.    Prepare 5ug of DNA into 2mL eppendorf.

2.    Add 100µL of protoplast suspension and 100 µL PEG solution. Mix by tapping the tube gently.

3.    Heat the mixture at 45⁰C for 5 min.

4.    Transfer the tube to room temp and leave for 5 min with occasional gentle mixing.

5.    Add 100µL of 8.5% mannitol solution. Gently invert the tube to mix. Wait at least 1min.

6.    Repeat step 5 five times.

7.    Add 300µL 8.5% mannitol solution. Invert gently to mix. Wait at least 1 min.

8.    Repeat step 7 four times

9.    Centrifuge at 100-200g for 5 min at room temp.

10. Discard the supernatant and resuspend the pellet in 500µL of 8.5% mannitol solution. Determine the quantity if molten PRMT needed (1volume of protoplast for every 2 volume of PRMT).

11. Add 1mL of molten PRMT. Pipette 1mL of the mixture gently but quickly onto each 90-mm plates containing PRMB and overlaid with cellophane.

12. Incubate the plates in continuous white light for 5 days at 25⁰C.

13. Transfer cellophane and regenerating protoplast to Knop medium containing the appropriate antibiotic to select for transformants.