Transformation HT-115

1
Transformation HT-115

• James Caldwell High School

2
Grow ON, shaking at 37 C.
Add 3 ml of LB Amp to each tube.
Transformation HT-115
Incubate on ice for 10 min.
Get 16 sterile test tubes and label them with
your plasmid name. EX AB-1, AB-2. Place the
tubes in ice.
Day 1
Add 3 ul of the plasmid DNA to the corresponding
tubes.
Add 15 ul to each culture tube.
Get a frozen sample of HT-115 bacteria. Thaw the
cells on ice.
Resuspend the HT-115 cells.
3
Label 16 microfuge tubes with the plasmid names
and HT-115
Day 2
Use a P-1000 to aliquot 400 ul of 80 sterile
glycerol to each tube
Store the tubes in your HT-115 freezer box
Resuspend the ON cells and add 400 ul of the
cells to the corresponding tube.
Label a cardboard storage box.
4
Why?!?!?!?!?!?!?!?!?!?!?!??!?

• DH5a is not able to synthesize double-stranded
  RNA.
• Therefore we use HT-115 to introduce double
  stranded RNA into the worms in order to block
  specific gene expression.
• The DNA of interest has to be transformed into
  HT-115 and fed to the worms.

5
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Get 16 sterile culture tubes and label.
• EX AB-1, AB-2, AB-3

6
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Place the tubes on ice.

7
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Get a frozen sample of COMPETENT HT-115 bacterial
  culture and gently thaw ON ICE.
• Competent bacteria can uptake a plasmid through
  their cell wall and membrane.

8
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Tap the tube to resuspend the cells.

9
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Add 15 ul of the bacterial cells to each
  corresponding tube.

10
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Add 3 ul of the plasmid DNA to the corresponding
  tube with competent cells.
• Incubate ON ICE for 10 minutes.

11
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Add 3 ml of LB AMP to each tube and grow
  overnights.
• Shaking at 37 C

12
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Label 16 microfuge tubes with the plasmid names
  and HT-115.

13
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Use a P-1000 to transfer 400 ul of 80 sterile
  glycerol to each tube.

400 ul
14
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Resuspend the overnight cells and add 400 ul of
  the cells to the corresponding tube.

400 ul
15
How?!?!?!?!?!?!?!?!?!?!?!?!?

• Label a carboard storage box with your initials
  and school name and HT-115 freezedowns.
• Store the box in the Freezer.

16

• Where do Waksman vampires live?

17
Transformania
HAHAHA !
18
THE END