Injection of C. elegans

 

<center> <b><font>Injection of </font> <i><font>C. elegans</font> </i> </b></center>

 

 

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Prepare injection pads

• line up three 22x50 mm coverslips
• place drop of molten 2% agarose on each
• quickly place slide on top of the agarose and wait 20 seconds (see figure)

 

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• carefully separate coverslips from slide so that the agarose pad remains on the coverslip and dry (overnight at room temp or 15 minutes on 65 degree heat block)
  ~undefined_hint~I_press_thumb_on_coverslips_before_dripping_the_agarose_~F_the_agarose_tends_to_stick_to_the_~4dirty~4_surface_of_the_coverslip_instead_of_the_slide~2~1~0~0~K~Hul~M~2~1~0~K~Hli~M~2~1~K~Hul~M~2~1~Kh2~M~2~1~0Load_needle_with_RNA~K~Hh2~M~2~1~Kul~M~2~1~0~Kli~M~2~1~0~0pull_needles_~ANarishige_puller_settings~I_4~H8~H10~B_from_borosilicate_glass_capillaries~K~Hli~M~2~1~0~Kli~M~2~1~0~0spin_down_RNA_prep_to_pellet_any_debri_~Afull_speed_5_minutes~B~K~Hli~M~2~1~0~Kli~M~2~1~0~0load_0.5_ul_RNA_onto_end_of_needle_and_allow_it_to_fill_the_tip_by_capillary_action~K~Hli~M~2~1~0~Kli~M~2~1~0~0mount_needle_on_micromanipulator~K~Hli~M~2~1~0~Kli~M~2~1~0~0break_needle_tip_until_RNA_flows_out_of_the_tip~K~Hli~M~2~1~K~Hul~M~2~1~Kh2~M~2~1~0Mount_worms_for_injection~K~Hh2~M~2~1~Kul~M~2~1~0~Kli~M~2~1~0~0place_drop_of_heavy_mineral_oil_on_pad~K~Hli~M~2~1~0~Kli~M~2~1~0~0pick_up_~4younger~4_adult_with_platinum_wire_pick~K~Hli~M~2~1~0~Kli~M~2~1~0~0transfer_worm_to_drop_of_oil_and_allow_the_worm_to_touch_the_pad_~Ahead_or_tail~B~K~Hli~M~2~1~0~Kli~M~2~1~0~0upon_touching_the_pad~E_the_worm_will_begin_to_stick_and_you_can_slide_the_pick_out_from_under_the_worm_so_that_the_worm_lies_relatively_flat_and_extended~K~Hli~M~2~1~K~Hul~M~2~1~Kh2~M~2~1~0Inject_worm~K~Hh2~M~2~1~Kul~M~2~1~0~Kli~M~2~1~0~0set_up_injector~I~2~1~0~0~Kul~M~2~1~0~0~0~Kli~M~2~1~0~0~0~0turn_on_micro~Finjector~K~Hli~M~2~1~0~0~0~Kli~M~2~1~0~0~0~0open_valve_on_pressure_tank~K~Hli~M~2~1~0~0~0~Kli~M~2~1~0~0~0~0open_valve_of_pressure_regulator~K~Hli~M~2~1~0~0~0~Kli~M~2~1~0~0~0~0set_injection_pressures~2~1~0~0~0~0~Kul~M~2~1~0~0~0~0~0~Kli~M~2~1~0~0~0~0~0~0line_pressure~I_approx_4500_hPa~K~Hli~M~2~1~0~0~0~0~0~Kli~M~2~1~0~0~0~0~0~0injection_pressure~I_900_~F_1200_hPa~K~Hli~M~2~1~0~0~0~0~0~Kli~M~2~1~0~0~0~0~0~0back_pressure~I_80_hPa~K~Hli~M~2~1~0~0~0~0~K~Hul~M~2~1~0~0~0~K~Hli~M~2~1~0~0~K~Hul~M~2~1~0~K~Hli~M~2~1~0~Kli~M~2~1~0~0place_mounted_worm_on_scope_~A10X_lens~B_and_orient_the_worm_at_a_30_degree_angle_to_the_needle_direction~K~Hli~M~2~1~0~Kli~M~2~1~0~0switch_to_higher_magnification_~A40X_lens~B_and_bring_gonad_into_focus~2~1~0~0~Kul~M~2~1~0~0~0~Kli~M~2~1~0~0~0~0honeycomb_pattern_is_the_surface_view_~F_lumen_view_has_granular_material_in_the_core~K~Hli~M~2~1~0~0~K~Hul~M~2~1~0~K~Hli~M~2~1~0~Kli~M~2~1~0~0move_needle_into_the_same_focal_plane~K~Hli~M~2~1~0~Kli~M~2~1~0~0press_needle_into_the_gonad_~F_the_gonad_will_deform_around_the_needle~K~Hli~M~2~1~0~Kli~M~2~1~0~0tap_side_of_the_stage_sharply_to_cause_the_needle_to_puncture_the_gonad_wall~K~Hli~M~2~1~K~Hul~M~2~1~Kp~M~2~1~0 
  <center> </center>

   

  • depress peddle to begin filling gonad with solution
  • stop filling when gonad expands along entire length but before it "pops" by depressing peddle second time
  • pull needle out of gonad
    • clear sticky debri from needle by applying line-pressure to cause solution to flow from tip
  • inject other gonad in similar manner

  Recover worms

  • place drop of egg-salts on worm to "rehydrate" (worm should begin swimming) and allow about five minutes recovery at room temp
  • once the worm is swimming well, transfer to OP50-seeded plate with mouth-pipette
  • passage worm at regular time intervals to determine timing of onset of RNA-interference effects

  Hints

  • good needles are the key ...... and largely dependent on your luck when breaking the tip
  • break needle by gently pressing tip against 18x18 mm coverslip mounted on drop of oil on 22x50 mm coverslip
  • if the worms move too much on the pad try drying the pads on heat block immediately before using
  • can do multiple injections on single pad by using small drops of oil at multiple spots: inject worm and recover it in egg salts while mounting and injecting a second worm in neighboring drop, when second is complete, transfer the recovered first to plate, etc ....
  • can inject multiple worms within the same drop of oil - just don't do too many that it takes too long and the worms dessicate before you can recover them
  • recovery time longer that 5-10 minutes usually not necessary - even non-swimming worms will recover after transfered to seeded plate

  References

  • Fire, A., et al., (1998) Nature 391:806-811
  • Mello, C. C., et al., (1991) EMBO 10:3959-3970