REAGENTS

Agar (500 g; Fisher Cat.# S70213)

GenePure LE Agarose (ISC Bioexpress Cat.# E-3120-500)

AvrII (NEB Cat.# R0174S)

BamHI (NEB Cat.# R0136S)

BglII (NEB Cat.# R0144S)

Gatway LR Clonase II Enzyme Mix (Invitrogen Cat.# 11791-020)

DAPI (Fisher Cat.# ICN15757401)

1 mm Electroporation cuvettes (Fisher Cat.# BTX610)

2 mm Electroporation cuvettes (Fisher Cat.# BTX620)

pENTR/D-TOPO lit with TOP10F (20 rxn.; Invitrogen Cat.# K2400-20)

GoTaq Green Master Mix (Promega Cat.# M7122)

HindIII (NEB Cat.# R0104S)

KpnI (NEB Cat.# R0142S)

1 kb Ladder (NEB cat.#N3232S)

T4 DNA Ligase (NEB Cat.# M0202S)

Montage PCR Single Sample PCR Cleanup (Millipore Cat.# UFC7PCR50)

NheI (NEB Cat.# R0131S)

NotI (NEB Cat.# R0189S)

100 mM dNTP set (10µmol each; Promega Cat.# U1330)

Paclitaxel (5 mg Fisher Cat.# ICN19353205; 25 mg Fisher Cat.# ICN19353225)

Paromomycin Sulfate (Sigma Cat.# P5057-5G)

PCR Cloning Kit (QIAGEN Cat.# 231222 or 231122-without cells)

0.2-mL Flat Top PCR Tubes (1000/bag; BIO-RAD Cat.# TFI-0201)

Pen/Strep/fungizone (Fisher Cat.# SV3007901)

Phusion High-Fidelity DNA polymerase (100U; NEB Cat.#F-530S)Proteose

Peptone (500g, Fisher Cat.# DF0122-17-4)

TopTaq DNA Pol. (250U; QIAGEN cat.#200203)

QIAEX II Gel Extraction Kit (QIAGEN Cat.# 20021)

QIAprep Spin Miniprep Kit (50 ct; Qiaprep Cat.# 27104)

AMV Reverse Transcriptase (Promega Cat.#M5101)

RNase Inhibitor (NEB Cat.#M0307S)

SalI (NEB Cat.# R0138S)

SacI (NEB Cat.# R0156S)Sodium Acetate (Fisher Cat.# S209-500)

SpeI (NEB Cat.# R0133S)

Freezer Storage Boxes (12 ct, Fisher Cat.#11-678-24A & 11-678-24C-

Dividers).

Tris Base (Fisher Cat.# BP152-1)

Wooden Stick Applicators (Fisher Cat.# 19086333)

XbaI (NEB Cat.# R0145S)

XhoI (NEB Cat.# R0146S)

Yeast Extract (Fisher Cat.# DF0127-17-9)

COMMON RECIPES

Ampicillin Stock

Prepare stock at 100 mg/ml (5.0g ampicillin sodium salt to 50ml in dH2O). Filter sterilize, prepare 5ml aliquots in sterile falcon polypropylene tubes, # each tube in series.

0.5 M HEPES stock pH7.5

For 100 ml, weigh out 13.01g and add to 75 ml dH2O, adjust pH w/ 12N HCl. Bring to volume. Filter sterilize. Store on bench. When making working solution (ie 10mM), this must be autoclaved before using w/ Tetrahymena.

Stock IPTG (200mg/ml or 0.8M)

Dissolve 2 grams of IPTG in 8 ml autoclaved water. Adjust volume to 10 ml with autoclaved water and filter sterilize using 0.2µm filter. Aliquot into 1.5 ml tubes and store at -20C

Kanamycin Stock

Prepare stock at 50 mg/mL (2.5g ampicillin sodium salt to 50ml in dH2O). Filter sterilize, prepare 5ml aliquots in sterile falcon polypropylene tubes, # each tube in series.

LB Media

Broth:

10g/l bacto-tryptone

5g/l Yeast Extract

10g/l NaCl

ForPlates:

-add 15 g bacto-agar

To 2 liter flask, add one 950 mL dH2O, mix solution, Then bring up to 1 liter. Make foil cap, mark with autoclave tape, autoclave for 30-45 mins. on liquid cycle.

10 mM dNTPs

Add 10 µL of each dNTP (dATP, dCTP, dGTP, and dTTP) together then add 60 µL of RNase/DNase free water. Then aliquot 10 µL to 0.5-mL tubes and freeze at -20°C.

2% PPYS20% PPYS (frozen stock)

20.0 g Proteose peptone200g Proteose peptone

2.0 g Yeast extract20 g Yeast extract

30.0 mg Sequestrine0.3 g Sequestrine

Bring to 1 L. Add the solids with the water stirring vigoursly. Spin at 7000 for 20 minutes. Store frozen (-20 C) in 200 ml aliquots.

SOB media

To 950 mL of deionized H2O add:

20 g Tryptone

0.5 g Yeast Extract

0.5 g NaCl

Then stir until dissolved. Add 2.5 mL of 1 M KCl and adjust pH to 7.0 with 5 N NaOH (~0.2 mL). Then bring volume up to 1.0 L and sterile by autoclaving. Just before use add MgCl2 (5 mL/1 Liter or 50 µL/10 mL). Note: Add 5ml 2M MgCl2 / 1 L SOB just before use.

SOC Media

Same as SOB media but just before use after autoclaving add 40% Glucose to final concentration of 0.4 % (10 mL/ 1 liter or 100µL/ 10 mL) when adding MgCl2 (5 mL/1 Liter or 50 µL/10 mL). If you do not use all of the media it can be stored at -20°C.

Sucrose Lysis Buffer for miniprep (50ml)

[Stock] amount [final]

sucrose4g8%

Triton x-1000.25ml0.5%

(very viscous, weigh out)

0.5M EDTA5.0ml50mM

1M Tris pH8.00.5ml10mM

H20enough to make 50ml

TAE 50x Gel Buffer

For 1 liter

242.0 g Tris Base

57.1 ml Glacial Acetic Acid

100.0 ml 0.5 M EDTA pH 8.0

Fill to volume with ddH20.

TBE (5x) Gel Buffer

For 2 litersfinal (5x) concentration

108 g Tris Base (Trizma)0.45M

54 g Boric Acid0.45M

20 ml O.5M EDTA ph 8.00.01M

Fill to volume with ddH20. (pH ~ 8.3)

TE (Tris-EDTA pH 8)

10mM Tris pH 8.0

1 mM EDTA pH 8.0

500 µl 1M Tris pH 8.0 + 100 µl 0.5M EDTA + 49.4 ml water/

Make 50 mls (or less) and store in a 50 ml Corning tube on bench.

Stock X-gal (20mg/ml)

Dissolve 200mg X-gal in 10 ml of dimethylformamide, than aliquot into 1.5 ml tubes and wrap in foil to prevent damage by light and store at -20C.