Wargo Laboratory

10x running buffer

• 30g Tris
• 144g Glycine
• 10g SDS
• Fill to 1L ddH₂O

300.19 medium

300.19 are an Abelson leukemia virus transformed murine pre-B cell line derived from Swiss Webster mice (H-2d). They are surface Ig negative and negative for B7-1, B7-2, and CD40. They express B220. They are non-adherent single cells. Pass by aspirating 99% of the cells every 2 or 3 days and adding new media. Pass to a new flask every 1 week or so to avoid selecting adherent cells. If the media turns very yellow, the cells may all die and be unrecoverable. They absolutely need the mercaptoethanol and will die in about 3 days without it. They grow very quickly.

• 440mL RPMI-1640
• 50mL fetal bovine serum (FBS)(10%)
• 5mL pen/strep (1%)
• 0.83mL of 10mg/mL gentamycin (optional, 15ug/mL)
• 5mL glutamine or glutamax (1%)
• 3.5uL 2-mercaptoethanol

300.19 hPDL1 blasticidin medium

• 50mL 300.19 medium
• 28uL blasticidin at 10mg/mL (or 56uL at 5mg/mL)

5x SDS loading buffer

• 10% SDS
• 30% glycerol
• 0.02% Bromophenol Blue
• 250mM Tris-HCl

Make about 10mL as a stock. Aliquot 950uL of the stock into an Eppendorf tube with 50uL 2-mercaptoethanol.

Blocking solution

• 5% BSA (for primary ab in western blots)
• 100mL TBST
• 5g Bovine Serum from Albumin (BSA)
• 200uL 10% sodium azide
• 5% milk (for secondary ab in western blots)
• 100mL TBST
• 5g instant milk

Cell culture medium

• 445mL DMEM
• 40mL heat-inactivated FBS
• 5mL pen/strep
• Pass through sterile filter

Cell lysis buffer

• 20mM Tris-HCl (pH 7.5)
• 150mM NaCl
• 1mM Na EDTA
• 1mM EGTA
• 1% Triton-X
• 2.5uM Na pyrophosphate

Inhibitors to add:

• 1mM beta glycophosphate
• 1mM Na VO4
• 1ug/uL leupeptin
• 1mM PMSF

Citrate buffer solution

• 2.94g sodium citrate
• 1000mL H₂O
• HCl to pH 6.0

Co-immunoprecipitation buffer

For apoptotic proteins, extract in CHAPS buffer (gentler detergent than RIPA):

• 5mL 10% CHAPS (10g/100mL in H₂O)
• 1.37mL 5M NaCl
• 100uL 0.5M EGTA
• 100uL 0.5M EDTA
• 250uL 1M Mg₂Cl₂
• 1mL 1M Tris (pH 7.5)
• H₂O to 50mL

Digestion buffer

For genomic DNA extraction

• 100mM NaCl
• 10mM Tris-HCl, pH 7.9
• 25mM EDTA, pH 8.0
• 0.5% SDS

Freezing medium

• 9mL sterile heat-inactivated FBS
• 1mL DMSO

Freeze cells slowly using the Mr. Frosty.

RIPA buffer

• 50mL 1M Tris-HCl (pH 8.0)
• 30mL 5M NaCl
• 10mL Np-40
• 5g Na deoxycholate
• 10mL SDS
• ddH₂O to 1000mL

To prepare RIPA buffer with PhostSTOP and protease inhibitor:

• 10mL RIPA buffer
• 2 tablets of "Complete" protease inhibitor
• 1 tablet PhosSTOP
• 200mL 50x cocktail
• Vortex and freeze

Better RIPA buffer (500mL):

• 3.02g Tris-HCl (pH 7.4)
• 4.38g NaCl
• 5mL Np-40
• 2.5g Na deoxycholate
• 0.5g SDS
• ddH₂O to 500mL

10x TBS

• For 0.5M Tris
• 500mL of 1M or 60.6g Tris-HCl
• 300mL of 5M or 87.6g NaCl
• Distilled water to 1000mL
• HCl to pH 7.6
• For 0.2M Tris
• 24.23g Tris-HCl
• 80.06g NaCl
• Distilled water to 1000mL
• HCl to pH 7.6

Wash buffer (TBST)

• 100mL 10x TBS
• 1mL TWEEN 20
• ddH₂O to 1000mL

TIL medium

• 40mL cell culture medium, using AB human serum in the place of FBS
• 200uL Fungizone
• 1000CU/mL IL-2
• 40uL Cipro

Transfer buffer

• 5.82g Tris
• 2.93g glycine
• 0.375g SDS
• 200mL methanol
• ddH₂O to 1000mL