Biomod/2011/UTAustin/Hook'em Hybridizers:Methods

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Methods

UT-BIOMOD protocols

  1. 0.5M EDTA pH 8.0 with NaOH pre-calculated (500 mL) (from OWW)
    93.05g of Na2·EDTA·2H2O (FW 372.2)
    10.14g of NaOH (FW 40)
    500 ml H2O
  2. 10X TBE (200ml) (From Jared Ellefson)
    (Note:Make it fresh now b/c it precipitates, this is enough for both the 20% and dilution)
    21.6 grams Tris base
    11 grams Boric Acid
    8mL 0.5M EDTA
    adjust to 200mL w/ H2O
  3. 20% Acrylamide with 7M Urea in 1x TBE (1L) (Jared Ellefson)
    Add one 500mL bottle of 40% 19:1 Tris
    Add 100mL of 10X TBE
    Add 420grams Urea
    mix with stirbar until dissolved (~2hrs)
    adjust to 1L H2O
    Filtered with .45uM
  4. Dilution Buffer with 7M Urea in 1x TBE(1L) (Jared Ellefson)
    Add 100mL of 10X TBE
    Add 420grams Urea
    mix with stirbar until dissolved (~2hrs)
    adjust to 1L H2O
    Filtered with .45uM
  5. 10% Acrylamide (native) in 0.25x TBE, 5% Glycerol (1L) (Xi Chen)
    Mix:
    • 40% Acrylamide (19:1) 250 mL
    • 1x TBE 250 mL
    • 100% Glycerol 50 mL
    • Water 450 mL
    Filter through .45 μm filter.
  6. 1x TBE (10 L) (modified from Tony Hwang)
    First dissolve 108 g Tris Base and 55 g Boric Acid into ~ 1 L H2O in a 1 L container, using a stir bar. Add 40ml of 0.5M EDTA (pH 8.0) to the roughly 1 L dissolved solution.
    While stirring, fill 10 L carboy to the "9 L H2O line" with DI water.
    Once the ~ 1 L concentrated TBE has dissolved, simply pour it all into the 10 L carboy, fill to the "10 L H2O line" with DI water.
  7. 1M MgAc2 (to be added later)
  8. Making/Running 10% Native gel (0.25x TBE, 10 mM MgAc2) (Xi Chen)
    Making gel:
    In a 50 mL conical tube, Mix 50 mL of 10% Acrylamide (native) in 0.25x TBE, 5% Glycerol and 0.5 mL of 1M MgAc2
    Add 500 μL 10% APS, 50 μL TEMED, mix by gently inverting the conical tube 5 to 10 times -- avoid creating air bubbles.
    Pour gel solution to glass plate assembly, apply comb, wait for gel to solidify.
    Running gel:
    Prepare 1 L of 0.25x TBE + 10 mM 1M MgAc2 by mixing 250 mL of 1x TBE, 750 mL of water, and 10 mL of 10 mM 1M MgAc2 -- This is your gel-running buffer.
    Prepare sample by mixing DNA sample with 1/5 V of 6x ThreeColor Dye
    Load sample w/ dye in wells, run gel at 250V w/ fan, for 5 to 8 hrs.

UT-BIOMOD lab rules

  1. Wear gloves at all time in the lab (never in the office). There are two reasons for doing this. First, it protects you from toxic reagents that had been accidentally spilled on the surface of benches, containers, equipments, keyboards of the computers that control equipments, etc. Second, it projects the reagents from contamination (especially RNase) that's carried on your skin.
  2. Isolate glove-free zones where you want to touch things with bare hands. You may want to designate an island on your bench to put lab notebooks, laptops, or other objects that you might touch outside the lab. Remember to take off gloves when you touch things in this island. You might as well establish a habit of where on drawers, doors, freezer handles, etc. you touch with gloves, and where without gloves. Ideally, the set of objects you touch with gloves and that without gloves should not intersect.
  3. Label ALL reagent containers. Enough information should be written on the label so you, and other people, know who made it, when and how it was made, what special attention should be taken, etc. We label the word "BIOMOD" on everything we use to separate our stuffs from other Ellington Lab stuffs. You initial should follow "BIOMOD", e.g. "BIOMOD-XY" or "BIOMOD(XY)". Let's standardize our naming convention: If the label is "BIOMOX(XY)", it means the person whose initial is XY made this reagent for BIOMOD team; everybody in the team can use it; and if this reagent runs low we should notify this XY person. If the label is "BIOMOD-XY", it means only this XY person can use this reagent.
    Some general and reagent-specific information that should be written is detailed below:
    1. Name and concentration of the reagent (mandatory for all reagents). Both standard (e.g. IUPAC) nomenclature and common name, both full name and acronym are acceptable, as long as it is unambiguous.
    2. Your initial (mandatory for all reagents)
    3. The date on which the reagent was made (mandatory for all reagents). This together with the initial allows one to trace back how the reagent was made on the lab notebook of the person who made it.
    4. Sterilization method (mandatory for stock solutions with > 10 mL volume). Typical sterilization methods include autoclave and filter. Preferably, you should add the parameter of the sterilization, e.g. temperature and duration of autoclave (typically 121 oC for 20 min) and pore size of the filter (typically 0.22 or 0.45 micron).
    5. Preparation method and purity (highly recommended for small-volume reagents prepared in the lab, including DNA strands and complexes). This is typically 'gel-purified', 'native gel-purified', 'HPLC purified'. You can also use your own acronym system as long as you can read it and keep the system consistent. This is sometimes important because the writing area on tubes is usually small.
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