Bridges Lab Protocols - User contributions [en]

Preparing Cell Lysates

2009-05-11T15:42:29Z

Darlanda:

Materials

RIPA Buffer (for 10mL lysis buffer)

Tris pH7.4 50mM 500uL

Na Deoxycholate 0.25% 250uL

NP-40 1% 1mL

NaCl 150mM 371uL

EDTA 1mM 20uL

NaVO3 1mM 100uL

NaF 1mM 20uL

Protease Inhibitors 1 tab

Basic Protocol
# Stimulate cells if necessary
# Wash cells 2x1mL with ice cold PBS -/- and aspirate
# Add 200uL RIPA buffer and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4C to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS
# Load gel

Preparing Cell Lysates

2009-05-11T15:41:51Z

Darlanda:

Materials

RIPA Buffer (for 10mL lysis buffer)
Tris pH7.4 50mM 500uL
Na Deoxycholate 0.25% 250uL
NP-40 1% 1mL
NaCl 150mM 371uL
EDTA 1mM 20uL
NaVO3 1mM 100uL
NaF 1mM 20uL
Protease Inhibitors 1 tab

Basic Protocol
# Stimulate cells if necessary
# Wash cells 2x1mL with ice cold PBS -/- and aspirate
# Add 200uL RIPA buffer and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4C to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS
# Load gel

Preparing Cell Lysates

2009-05-11T15:40:01Z

Darlanda:

Materials
RIPA Buffer (for 10mL lysis buffer)
Tris pH7.4 50mM 500uL
Na Deoxycholate 0.25% 250uL
NP-40 1% 1mL
NaCl 150mM 371uL
EDTA 1mM 20uL
NaVO3 1mM 100uL
NaF 1mM 20uL
Protease Inhibitors 1 tab

Basic Protocol
# Stimulate cells if necessary
# Wash cells 2x1mL with ice cold PBS -/- and aspirate
# Add 200uL RIPA buffer and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4C to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS
# Load gel

Splitting Cells

2009-05-06T15:30:53Z

Darlanda:

==Materials==
*Media (L1-FBS for 3T3-L1, COS-FBS for others): Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM
*PBS -/-
*0.05% Trypsin

==Protocol==
#Warm PBS and Media in water bath
#Wash cells twice with 10 mL (per 10 cm dish) PBS -/-
#Add 1 mL trypsin and sit in the hood
#Add 10 mL media to each new dish
#Check cells for trypsinization, and if necessary tap the cells
#Add 9 mL media to trypsinized cells
#Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X)
#Replace plates in the incubator

PCR Amplification of DNA

2009-05-05T16:02:40Z

Darlanda:

==Materials==
*Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water)
*Template – generally 1uL or less of a plasmid miniprep
*Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.

==Protocol==
*Use the following volumes per reaction
::*Buffer, 5 uL of 10X buffer (Dave's fridge)
::*Primers, 10uL of 1uM stock solution in water (both primers combined)
::*dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer)
::*Sterile water, 28 uL
::*Template 1 uL
::*Polymerase 1 uL (turbo pfu found in "enzymes" box in freezer)

*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''DAVETD''') as follows:
:#1 min at 94
:#30s at 65
:#2 min/kb at 72
:#30s at 94
:#30s at 63 then -0.5/cycle
:#2 min/kb at 72
:#Repeat steps 4-6 28 times
:#30s at 94
:#30s at 45
:#11 min at 72
:#Hold at 4 until ready
*Purify PCR product if necessary using Qiagen kit (Add 5x PB)