SequaGel (National Diagnostics cat# EC-833)
Temed (BioRad cat# 161-0800)
SafetyCoat: Baker cat# 4017-01
10% APS, Make a 10% w/v in DEPC treated H2O. Ammonium persulfate, Sigma
A7460. Keep in refrigerator, make fresh every week.
10xTBE: (890 mM) 216g Trisbase Fisher BP 152-5 (MW 121.14)
(890 mM) 110g Boric acid Fisher (electroph.gr) BP 168-500 (MW 61.83)
(20 mM) 40 mL 0.5M EDTA pH 8.0
H2O to 2L, check pH w. strip (take out and drizzle ~500 ul on
pH strip, ~pH 8.5, filter sterilize 0.2um, Fill 3 one liter bottles, mark level,
autoclave and bring up to level with H2O after cooling to RT
Formamide Loading buffer: 95% (v/v) formamide
0.09%( w/v) Bromophenol Blue
0.09% (w/v) Xylene Cyanol FF
Fluka Bromophenol Blue-Xylene Cyonol solid mixture cat no 18047, when reconstituted to 5 ml, ~ 0.5% each dye in Tris-borate-EDTA buffer pH 8.3. Add 1.39 ml DEPC H2O ~1.8% dye. Mix 95 uL Formamide with 5 uL 1.8% Bromophenolblue/Xylene cyanolFF
Note: can use less dye.
Hybond N+: Amersham RPN303B
ULTRAhyb-Oligo Hybridization Buffer: Ambion cat # 8663, keep in refrigerator
T4 Polynucleotide Kinase: NEB M0201S
Gamma 32P ATP, 6000 Ci/mmol, 150 mCi/mL PerkinElmer (cat# NEG035C005MC)
MicroSpin G-25 columns: Amersham 27-5325-01 (cellculture)
Wash buffer: 2xSSC/0.1%SDS
50 mL 20x SSC + 447.5 mL autoclaved H2O + 2.5 ml 20% SDS
This protocol has been put together with the help of many people from the McManus lab and surrounding labs at UCSF! Thanks for all of your help.