IGEM:MIT/2005/Tuesday, June 7th

MIT iGEM 2005 Laboratory "Bootcamp" Day 2 (run by Natalie Kuldell and Kate Bacon Schneider)

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