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 -20C
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 -20C.