May 18, 2016
By UNM CCC 
M.Fero — 1/17/2001 Final Concentration 5x SSC 25 mM NaPO4, pH6.5 5x Denhardt’s Solution 0.25 mg/mL Torula RNA 50% (v/v) Formamide Stock Solutions 20x SSC: 3 M NaCl, 0.3 M NaCitrate, pH7.0 50x Denhardt’s: 1% (w/v) BSA, 1% (w/v) PVP-40, 1% (w/v) Ficoll-400 10 mg/mL Torula RNA (boil to dissolve) 1 M NaPO4, pH 6.5 […]
Tags: Fero-DNA
May 18, 2016
Fluorometric Quantitation of RNA or DNA
By UNM CCC
M. Fero 11/05 • Adapted from Turner’s protocol (website) Materials DNA: Höchst 33258 (High range solution): (Invitrogen H1398 or Sigma B1155) 1µg/mL in 1x TNE Filter and store at 4ºC in an amber bottle. 10x TNE stock: 100 mM Tris, 2 M NaCl, 10 mM EDTA, pH7.4. Store at room temp. RNA: Ribogreen 200x (Invitrogen R-11491) detection reagent. Store dessicated at -20ºC. Turner Designs TD-360 […]
Tags: Fero-DNA
May 16, 2016
By UNM CCC
Measuring DNA concentration with the Nanodrop Spectrophotometer v.1 10-31-2011 A. AlKhinji Materials DNA in TE buffer (10 mM Tris pH8, 1 mM EDTA) TE buffer for blanking machine P20 micropipettor + tips H2O squeeze bottle and Kipwipes (at machine) Procedure Sign log book Login to computer: (Adjacent lab P.I./room) Launch software: ND-100 v.3.7.1 Wash the […]
Tags: Fero-DNA
May 16, 2016
By UNM CCC
M. Fero 11/2004 SYBRGreen Q-PCR in the ABI 7700 Materials ROX Passive Reference Dye 25 µM, 50x (Invitrogen, Cat. No. 12223-012) SYBR Green I, “10,000x” concentrate (Molecular Probes, Cat. No. S7563) SureStart Taq polymerase 5 U/µL (Stratagene, Cat. No. 600280) Solutions SYBR Green 1:100 dilution (50 µL SYBR Green concentrate, 5 mL DMSO, store aliquots […]
Tags: Fero-DNA
May 16, 2016
By UNM CCC
(M.Fero 9/2007) Reagents Lysis Buffer: (10 mM Tris pH8, 100 mM NaCl, 25 mM EDTA, 0.5%SDS), store at room temp. 10 mg/mL Proteinase K: 100 mg (Roche) + 10 mL buffer (10 mM Tris pH8.0, 20 mM CaCl2, 50% (v/v) glycerol), store at -20ºC. Lysis buffer + 1 mg/mL proteinase K: 50 mL Lysis buffer […]
Tags: Fero-DNA
May 16, 2016
By UNM CCC
Materials Prehybridization Solution (10 – 50 mL). Mix by rotating at 37°C: Start with 50 mL Stark’s Solution (with 50% formamide) + 0.5% non-fat dry milk (0.25 gm) + 1% SDS (0.5 gm) + 8% dextran sulfate (4 gm, Sigma D-6001) 42°C Shaking Water Bath 100°C Heater block 32P-labled probe Use 10 – 20 ng […]
Tags: Fero-DNA
May 16, 2016
By UNM CCC
Notes Stocks should be handled with care to ensure that they are not contaminated with PCR product or plasmid DNA that might be prevalent in the laboratory. For this reason they are handled only in the pre-PCR area with gloves, aerosol resistant tips, and DNAse/contamination-free solutions. Oligo concentrations are typically expressed in terms of moles, […]
Tags: Fero-DNA
May 16, 2016
By UNM CCC
Materials Electroelution gel box and elution block 0.25x TBE buffer 5 M KOAc with BPB (bromphenol blue) 100% EtOH 70% EtOH T.E. buffer (10 mM Tris, 1 mM EDTA, pH 8) Procedure Run your DNA fragment on a preparative agarose gel Add 350 mL of 0.25x TBE buffer into electroelution box. Buffer should cover the […]
Tags: Fero-DNA
May 16, 2016
AML Sample Genomic DNA Preparation
By UNM CCC
Solutions PBS Cell lysis buffer: 10 mM Tris pH8, 100 mM NaCl, 25 mM EDTA, 0.5% (w/v) SDS, 0.5 mg/mL proteinase K Fixative: 1 volume glacial acetic acid + 3 volumes methanol Trizol reagent 75% EtOH made with RNAse free H2O 100% EtOH Phenol: Buffered to pH=8 with Tris. Chloroform: Stabilized with isoamyl alcohol. T.E.: […]
Tags: Fero-DNA
May 16, 2016
By UNM CCC
(Ref: Molecular Cloning A8.12) The major considerations in using alcohol to precipitate DNA are: Temperature: -20°C is optimal, but 0°C can be used for >20 ng/mL Amount: For small amount of DNA (<100 ng, i.e. too little to reliably see a pellet) the use of glycogen (1 µL of a 20 mg/mL stock, (Roche 10901393001) […]
Tags: Fero-DNA
May 16, 2016
A/B/C Random Amplification Protocol
By UNM CCC
Adapted from Bohlander et al. Genomics 13 (1992). (modified by D. Schubeler, L. Loo) Genomic DNA will be randomly primed with a sequence tagged oligonucleotide for 2 cycles. This will create random genomic products with a specific tag at both ends. These products will then be amplified and labeled with a sequence specific dye primer. […]
Tags: Fero-DNA
