Chang Lab Protocols

E.coli

High Yield DNA Sequence Prep
Boiling Lysis DNA Prep
Phenol Chloroform DNA extraction

Transformation of Chemically Competent Cells

C. elegans

C. elegans Injection Prep
C. elegans Injection Protocol

NGM and RNAi Plate Recipes

S. cerevisiae

H-type Synthetic Growth Media For Yeast
5-FOA Yeast Plates

• Make 2 ml LB overnight
• Fuge for 30sec in 1.5 ml Eppendorf Tubes
• Pour out Sup.
• Resuspend in 300 m l STET
• VORTEX
• Boil with cap for 2min.
• Fuge for 5min
• Scoop out snot with toothpick.
• Add equal Volume of 75% Isopropanol 2.5M NH4Ac
• VORTEX
• Spin 5min
• Keep Pellet, aspirate off sup.
• Wash with 500 m l 70-80% EtOH
• Aspirate of Sup.
• SpeedVac Dry resuspend in H2O or TE.

STET
0.1M NaCl
10 mM Tris-Cl (pH 8.0)
1 mM EDTA (pH 8.0)
5% Triton x-100

75% Isopropanol 2.5 M NH4Ac (40 ml)
10 ml 100% Isopropanol
30 ml 10M NH4Ac

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Injection Prep
From Eric Haag 7-8-03

For Tranformation:

• pRF4 (or other marker)
• test plasmid

Regular Repetitive Array (non germline expression)

• 100 ng/ul DNA (anywhere from 50-200 ng/ul)
• 50 ng/ul of each (test plasmid + marker)

Complex Array (for germline expression, more difficult)

• Add ~95 ng/ul Carrier DNA (see FLV Docs for preparation)
• Mix in 2-3 ng/ul each of LINEARIZED Marker + test plasmid

RNAi

• Make dsRNA using Ambion Kit (Megascript) (2-4 hour prep)
• Template: cut FLV vector construct to release T7-flanked insert
• Clean up RNA w/ phenol chloroform according to kit instructions
• Run small 2-3 ul sample on gel to check

• Mix in Distilled Water or TE
• Prior to injection spin down junk for one minute in fuge at top speed.

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Materials Needed :

• dsRNA or Plasmid in H2O or TE
• M9 Solution
• Mouth Pipeter
• 20 ul Pipetman
• Picker
• Pick Plate (Sticky OP50 makes everything a lot easier)
• Plate of worms (young adults are used for injection)
• OP50 seeded plates for injected worms
• Sharpie

• Use filament Capillaries capable of back filling
• Insert Needle under aluminum foil and clamp on both ends
• Push needle holder past last click (there is a little stop right before the last click)
• Check settings and cover the unit
• Pull needle (The pulled needle is usually the better of the pair)

• To make filler needle pull a 20 ul pipet capillary over an ethanol flame
• Pull thin and break with end of picker
• Break of another small 2 cm fragment for the Needle Breaker
• Place this small capillary on a slide and cover with Mineral Oil, place on Microscope

• Spin down your solution to remove any unwanted contaminants
• Insert Needle Filler into mouth pipeter
• Draw a small amount of solution up the needle filler (to the point where the needle returns to its original diameter)
• Back fill the Needle by inserting Needle Filler into the backside of the needle and mouth pipeting the solution in.
• Tap the Needle several times and check under a microscope for unwanted air bubbles

Manipulator

• Load the needle onto the manipulator
• With the microscope focus on the Needle Breaker slide
• Adjust the manipulator until needle point is over microscope light source
• Adjust the needle position until it is in the mineral oil by the capillary

FemtoJet

• Pi = inject pressure
• Ti = inject time
• C = constant pressure

• Set Pi to ~35 psi
• Break needle by sliding along Needle breaker very gently and check by pressing the inject button on the mouse
• Adjust the Pi and C until needle injects at proper pressure

• Get an agar padded slide and breath on it multiple times to moisten it
• Place a small drop of Mineral Oil over first pad
• Obtain plenty of sticky OP50 and pick up a worm
• Place worm gently on agar pad and drag to straighten

Injection

• Place worm on stage
• Pull needle gently out of needle breaker mineral oil by using the angle adjuster
• Center the worm in the microscope light and check orientation under 10x
• Lower needle into mineral oil and focus with worm
• Position needle and worm in center of view and switch to 40x
• Slowly work needle into gonad area (or gut for dsRNA) and inject
• Several injections might be needed to fill gonad, check for the liquid front after injection
• Gonad is the clearer honeycomb region (inject right below honeycomb region)
• Remove needle from worm
• Raise needle out of worm oil and lower it back into needle breaker oil so that the dsRNA doesn’t dry out in the needle

Worm Rescue

• Take worm slide and drop ~13 ul of M9 on the worm
• Worm should start squirming if alive
• Carefully rotate picker beside the worm until the current lifts the worm off the agar pad
• Hook the worm and transfer it to a seeded OP50 NGM plate
• Pray to God that the worm lives for more than an hour

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Purification of DNA from any reaction

Dilute Reaction mixture to 100 ul with water

• Phenol Extraction
• Add 1x volume equilibrated phenol (if from stock phenol is lower layer)
• VORTEX
• Spin 5 min.
• Transfer aqueous layer (top) into new tube
• Chloroform Extraction
• Add 1x volume Chloroform
• VORTEX
• Spin 2 min.
• Transfer top layer to new tube
• Ethanol Precipitation
• Measure Reaction Volume (should be around 90-100 ul) “original volume”
• Add ½ original volume 7.5M NH4Ac
• Add 3x original volume of 100% Ethanol
• Spin 15 min.
• Remove sup.
• Wash with 70-80% Ethanol
• Dry in SpeedVac

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High Yield DNA Sequence Prep

1. Incubate 4ml culture in TB overnight
2. Pellet cells in fuge
3. Resuspend pellet in 200 ul GTE (or STE, Solution #1)
4. Add 300 ul fresh .2N NaOH/1% SDS (Solution #2),
mix by inversion (NO VORTEXING) and place
on ice for 5 minutes
5. Add 300 ul 3M K Acetate (pH 5.2, Solution #3), Invert several times
6. Fuge for 10min rt transfer sup to new tube (no crap should remain)
7. Add 1 ul RNase Stock (20 ug/ml) and incubate at 37C for 15-20min
8. Extract twice w/ 400 ul Chloroform
- Add 400 ul Chloroform
- VORTEX, fuge for 2min
- Keep TOP LAYER

9. Precipitate DNA by adding an equal volume of 2-propanol, VORTEX, fuge at rt for 10min
10. Wash Pellet w/ 500 ul 70% EtOH
11. Dissolve pellet in 32 ul dHOH
12. Add 8 ul 4M NaCl, and 40 ul 13% PEG-8000
13. Mix thoroughly and place on ice for 20min, then fuge at 4C for 15min
14. Wash pellet w/ 500 ul 70-80% EtOH, dry pellet in speedVac and resuspend in 20 ul dHOH or TE.
15. If desired, check 1 ul on a gel

Solution #1
200 ml
1.8g glucose
4 ml EDTA (50X)
5 ml Tris pH 8
191 ml dHOH

Solution #2
2 ml
200 ml 2N NaOH
200 ml 10% SDS
1600 ml dHOH

Solution #3
100 ml
11.5 ml glacial acetic acid
28.5 ml dHOH
60 ml 5M K-Acetate (29.55 g. K-Acetate in 60 ml dHOH)

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Invitrogen Library Efficient DH5 a Competent Cells

• Place appropriate number of Falcon 17x100 mm polypropylene tubes on ice
• Turn waterbath to 42 ° C
• Thaw Library Competent Cells on Ice.
• Aliquot 100 ul Competent cells into each chilled Falcon Tube.
• IMMEDIATLEY RE-FREEZE competent cells in ethanol+dry ice for 5 minutes then return to -80 ° C. no liquid nitrogen.
• Aliquot ~1 ul DNA from ligation into each Falcon Tube and let incubate on ice for 30 minutes.
• Heat-shock cells for 45 seconds in the 42 ° C water bath.
• Place on Ice for 2 minutes
• Add 900 ul of rt SOC Medium (provided in kit)
• Shake at 37 ° C for ~1 hour.
• Plate Cells on appropriate plates for o/n storage at 37 ° C.

Invitrogen Sub-cloning Efficient Chemically Competent Cells

• Place appropriate number of 1.5 ml tubes on ice
• Thaw Sub-cloning efficient cells on ice
• Aliquot 50 ul sub-cloning efficient cells into each 1.5 ml tube
• IMMEDIATLEY RE-FREEZE competent cells in ethanol+dry ice for 5 minutes then return to -80 ° C. no liquid nitrogen.
• Aliquot 2 ul DNA from ligation into 1.5 ml tubes containing competent cells
• Gently mix together
• Incubate on ice for 30 minutes
• Heat shock at 37 ° C for 20 seconds (use water bath)
• Immediately after add 1 ml room temp LB
• Place back on Ice for 2 minutes
• Shake at 37 ° C for ~1 hour
• Plate cells on appropriate plates

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H-type Synthetic Growth Media For Yeast

1. Add:

2. Adjust pH to 5.6 with NaOH (one drop at a time).

• pH step for plates is required for optimum transformation efficiency and it also makes the plates harder, it is not necessary for liquid media. You do not pH 5-FOA plates either.

3. Add:

4. Autoclave 20-30 minutes.

• Over autoclaving can result in soft plates

5. After autoclaving while media is cooling add:

H-type 20x Amino Acid Mix:

This is a 20x stock of amino acids. To make various split-ubiquitin plates (example: SD-H, SD-HT, SD-U, SD-MU, SD-MHU) make a 20x stock of the lower amino acids and then make individual 20x stocks for uracil, L-tryptophan, L-histidine, and L-methionine so that they can be added in appropriate combinations.

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5-FOA Yeast Plates

1. Add:

2. Let mix thoroughly by spinning.

3. While spinning add 1.0 g 5-FOA (US. Biological Cat# F5050) to 400 ml dH 2O for 1 Liter of plates or 0.5 g 5-FOA to 200 ml dH 2O for 500 ml plates.

4. Microwave for 45 sec followed by spinning until completely dissolved.

5. May require repeated short microwaving to completely dissolve, but do not boil.

6. Filter sterilize 5-FOA solution.

7. Return to Solution A and add:

8. Autoclave for 25-30 min.

9. Let media cool and add:

9. Pour plates.

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