IGEM:MIT/2005/Miniprepping DNA, Agarose gels, RE digests

I. Miniprepping DNA

• Background Information:
  • method for separating plasmid DNA from other cellular components (chromosomal DNA, RNA, proteins, lipids)
  • need to do a number of things in any miniprep protocol
    • lyse cells (heat, sonication, detergents) (and remove RNA)
    • precipitate proteins/chromosomal DNA/lipids
    • concentrate (by column or precipitation) miniprepped DNA
    • wash miniprepped DNA (removes salts)
    • resuspend in useful buffer (water, TE)
• Exercise:
  • purify plasmid DNA (BioBrick type) using Qiagen spin column kit

II. Making/Pouring agarose gels

• Background Information:
  • agarose is derived from seaweed
  • solubilizes when heated (in buffer), then polymerizes upon cooling
  • forms a matrix with "pores" through which nucleic acids can travel
    • altering concentration of agarose in gel changes size of pores and resolution of gel (lower agarose concentration best for separation of larger molecules)
    • 1% (weight/volume) gel = 1 gram of agarose/100 mL of buffer
  • Use Ethidium bromide (slides between bases of nucleic acids, fluorescent when exposed to UV light) to visualize DNA on gel
    • can add to gel before pouring, or use in buffer
  • Buffer needed to make and run gel-->want to use the same buffer for both making and running gel-->we used TAE (Tris, acetate, EDTA)
  • "Comb" used to create "wells" (place to load samples)
• Exercise:
  • make and pour 1% agarose gel (50 mL volume)

III. Restriction Enzymes/Restriction digests

• Background Information:
  • Restriction enzymes are the bacterial cell "immune system"—protect cells from invaders (viruses)
  • REs cut at specific places within specific sequences-->most useful in molecular biology are Type II enzymes (which have palindromic recognition sites).
  • Cutting can leave "sticky ends" (DNA overhangs that can basepair with other, complementary overhangs) or "blunt" ends (no overhangs)
  • Action of RE is to break the phosphodiester bond joining two nucleotides in a DNA sequence--> action of enzyme leaves a 3' OH and 5' P that can be rejoined in a "ligation" reaction
  • RE, like all proteins, work in specific reaction conditions:
    • need buffer with proper salt concentrations and Mg+2
    • need DNA substrate
    • need enzyme (<10% of total volume)
    • need water
  • usually do 20 µL reactions for 30 minutes-1 hour for complete digestion (with excess enzyme)
  • Addition order is critical—want to always add enzyme last (enzymes are fragile, need to be kept properly folded to be functional)
• Exercise:
  • Gave students plasmid pSB1A3 (NK and KBS knew this was BioBrick vector of 2157 bp + ~900 bp insert),the RE enzymes EcoRI, SpeI, and PstI, and NEB2 (compatible buffer for all three REs).
  • Had students set up single digests with EcoRI and one of the other two, then the double digest (EcoRI/SpeI or EcoRI/PstI)-->ran for 60 minutes @ 37˚C
  • Students' job was to determine the size of the insert after running on agarose gel
  • Also would be used for purification of DNA from agarose/ligations/transformations later in the week