Reagents

Stock Solutions:

0.1M dTTP

0.1M dGTP

0.5M EDTA (pH 8.0)

300mM Tris-HCl (pH 7.4)

BSA (20mg/ml)

10% SDS

2.5M Tris (pH 8.0)

250mM MgCl₂

Chloramphenicol (20mg/ml)

10N NaOH

10% SDS

RNase A (10mg/ml)

Isopropanol

80% Ethanol

Pre-Hybridization Solution (Church Buffer)

To make 4L total:

While stirring, heat 400ml dH₂O on the hot plate until just warm (Caution! Do not heat too much or the BSA will aggregate)

Add 40g BSA (Sigma A-4503). This might take a couple hours to dissolve.

Make 2L of 1M Sodium phosphate solution

In 4L beaker with stir bar, measure 268g Na₂HPO₄·7H₂O

Dissolve in 1700ml dH₂O

Add 8ml of 85% H₃PO₄

Q.S. to 2L with dH₂O

Add to the phosphate solution while still stirring:

0.5M EDTA (pH 8.0) 8ml

SDS 280g (Wear face mask and scoop out carefully to measure. Extremely hydroscopic.)

Heat 800ml dH₂O to 65°C and add to the Phosphate/SDS solution to warm the buffer. This will help the SDS go into solution.

Add the 400ml dissolved BSA and bring the final volume up to 4L with warm distilled water

Pass through a coffee filter funneled into a 4L jug.

Solution O (100 ml)

2.5M Tris (pH 8.0) 50ml

250mM MgCl₂· 6H₂O 50ml

Add 50ml of both the sterilized solutions to an autoclaved bottle and mix. Store at 4°C.

Solution A (1028ml)

Solution O 1ml

2-Mercaptoethanol 18ml

0.1M dTTP 5ml

0.1M dGTP 5ml

Add all reagents to a 1.5ml tube and mix thoroughly. Aliquot of 40ml into 1.5ml tubes. Store at -20°C.

Solution B (250ml)

HEPES 119.15g

NaOH pH to 6.6

dH₂O Q.S. to 250ml

Measure out the HEPES into a beaker and dissolve in about 200ml water. Bring the pH to 6.6 with NaOH. Bring to a final volume of 250ml and filter sterilize. Store at 4°C.

Solution C (100ml) Final Concentration

300mM Tris-HCl (pH 7.4) 3ml 3mM

0.5M EDTA (pH 8.0) 40ml 0.2mM

dH₂O Q.S. to 100ml

Store at 4°C.

OLB Reagent

Solution A 40ml

Solution B 100ml

Solution C 60ml

Mix all three solutions together and keep on ice until used. Store at -20°C.

20X SSC

Trisodium Citrate 88.2g

NaCl 175.3g

dH₂O Q.S. to 1L

Measure out the Sodium Citrate and Sodium Chloride and dissolve in about 800ml water. Then add pellet of Sodium hydroxide and bring up to a final volume of 1L. Store at room temperature.

2X SSC/ 0.1% SDS

20X SSC 100ml

10% SDS 10ml

dH₂O Q.S. to 1L

1.5X SSC/ 0.1% SDS

20X SSC 75ml

10% SDS 10ml

dH₂O Q.S. to 1L

1.0X SSC/ 0.1% SDS

20X SSC 50ml

10% SDS 10ml

dH₂O Q.S. to 1L

Stripping Buffer

20X SSC 500ml

10% SDS 1ml

dH2O Q.S. to 1L

LB agar media (1L)

Bacto Tryptone 10g

Bacto Yeast Extract 5g

NaCl 10g

Agar 15g (USB #10654)

dH2O Q.S. to 1L

Chloramphenicol(20mg.ml) 1ml

Add about half the water to dissolve the ingredients in and then measure to the full volume with water. Place a stir bar into the bottle and close the media bottle. Sterilize by autoclave. When done autoclaving, loosen the lid without removing and then place on a magnetic stir plate and stir while allowing to cool until warm. Do not let the media cool too much or it will solidify. Add the chloramphenicol and stir briefly to mix in. Pour about 20 ml into each 15 X 100mm petri plate. If is does not cover the bottom completely agitate the plates slightly so the media fills the bottom. Do not touch again until completely cooled and solidified.

*When ready to use, we turn the plates over and divide the plate by drawing a line down the center and then taping each two consecutive clones from a computer printout over the line facing down.

LB storage media

(1L) Bacto Tryptone 10g

Bacto Yeast Extract 5g

NaCl 10g

Glycerol 75ml

dH₂O Q.S. to 1L

Chloramphenicol(20mg.ml) 1ml

Add about half the water to dissolve the ingredients in and then measure to the full volume with water. Sterilize by autoclave. When cooled to room temperature, add the chloramphenicol and mix in well. Aliquot 175ml of the media into each well of the 96-well plate (Corning 3799), requiring about 20ml total to fill each plate. We use the Q-fill2 (Genetix) automated dispensing system to fill all the plates in this procedure.

2X YT media

(1L) Bacto Tryptone 16g

Bacto Yeast Extract 10g

NaCl 5g

dH₂O Q.S. to 1L

Chloramphenicol (20mg/ml) 624ml

Add about half the water to dissolve the ingredients in and then measure to the full volume with water. Sterilize by autoclave. When cooled to room temperature, add the chloramphenicol and mix in well. Aliquot 1.2ml of the media into each well of a 96-deep well plate (Beckman 140504) requiring about 150ml for each 96-well plate filled.

GET/RNase buffer Add Final concentration

Glucose 2.25g 50mM

2.5MTris-HCl(pH 8.0) 2.5ml 25mM

0.5M EDTA (pH 8.0) 5ml 10mM

dH₂O Q.S. to 250ml

Weigh out the glucose and add to about half the volume of water to dissolve. Add the Tris and EDTA. Bring the final volume up to 250ml with water and pass through a sterilizing filter (Corning 430767). Store this at 4°C. For preparation of 2x 96-well plates, you will need to measure out 40ml of this buffer. To the 40ml, add 300ml RnaseA (10mg/ml) immediately before use.

NaOH/SDS Lysis Buffer This needs to be freshly prepared from the stock solutions before each use. Add Final concentration

ddH₂O 35.2ml

10N NaOH 0.8ml 0.2N NaOH

10% SDS 4ml 1% SDS

Measure out the water and then add the stock solutions in a 50mlconical tube. This will make 40ml total, enough for preparation of two plates. Do not chill this reagent or place on ice.

Potassium Acetate Buffer

KC₂H₃O2 (F.W. 98.14) 73.16g

Glacial Acetic Acid 28.75ml

dH2O Q.S. to 250ml

Measure out the Potassium Acetate and dissolve in about 200ml dH₂O. Add the Acetic Acid and bring up to the final volume of 250ml with dH₂O. Pass through a pass through a sterilizing filter (Corning 430767). Store this at 4°C.