From 2008.igem.org

Revision as of 22:27, 17 October 2008

Lab Protocols

In Progress

Overnights

Inoculating LB culture tubes:

   Add 5ul Amp50 or Amp100 to 5ml LB broth in tube.
   Using sterile inoculating loop, touch an isolated colony.
   Swirl loop through LB + amp broth.
   Incubate overnight at 37 C.

Minipreps

QIAprep Spin Miniprep Protocol

Isolating DNA from LB culture tubes:

   Add 2X 750ul broth from overnights into a 1.5ml tube.  Centrifuge for 1 min to pellet and aspirate liquid.
   Add 250ul Buffer P1 * and resuspend pelleted cells.
   Add 250ul Buffer P2 ** and invert tube 4-6 times.
   Add 350ul Buffer N3 and invert tube immediately 4-6 times.
   Centrifuge for 10 min.
   Apply supernatant to QIAprep spin column.
   Centrifuge for 1 min and discard flow through.
   Add 500ul Buffer PB, centrifuge for 1 min and discard flow through (only if high nuclease activity).
   Add 750ul Buffer PE, centrifuge for 1 min and discard flow through.
   Centrifuge for an additional 1 min.
   Place spin column in a clean 1.5ml tube.  Add 50ul Buffer EB, let stand for 1 min and centrifuge for 1 min.
   These may be stored at -20 C.

*stored at 4 C
**heat first in 42 C waterbath for 5 min

Digests

   Reaction mix:
   DNA                   5-15ul
   10X NEB 2 buffer      10% of total volume
   10X BSA               10% of total volume
   Enzyme 1              0.5ul                       ALWAYS KEEP ENZYMES ON ICE!
   Enzyme 2              0.5ul
   MQH20                 as need to bring up to total volume

   Add to PCR tubes and incubate at 37 C for 1-2 hrs.
   Liquify 2% agarose gel (for fragments <2 kbp) by heating in microwave.  
   Pour into gel mould - don't forget the well-maker!
   Add dye to PCR tubes such that 1/6 of total volume is dye. 
          (ie. 20ul digest mix + 4ul dye = 24ul total volume. 4/24 = 6)
   Load 10ul of DNA ladder into first well.
   Load 10-30ul of DNA digest (+dye) into the remaining wells.  NEVER MAKE YOUR GEL SYMMETRICAL!
   Add fresh TEB if DNA fragments in the gel will be excised.  Use "used once" TEB if not.
   Run at 110V for small gels or 140V for large gels for ~50 min.  KEEP AN EYE ON YOUR GEL!
   Soak the gel in ethidium bromide for 10-15 min.
   If you are excising a fragment: view the gel under low frequency UV light.
   If you are simply viewing the gel: view the gel under high frequency UV light.
   Save a copy of the gel and print off a picture.

   To excise a gel fragment:
   ENSURE YOU ARE WEARING A UV FACE SHIELD, GLOVES AND LONG SLEEVES.
   Using a razor blade, cut out the desired gel fragments.  Avoid excess agarose.  
   Place into labeled 1.5ml tubes.
   These may be stored at -20 C.

PCR

  Reaction mixture:
  10X PCR 20 buffer   2.5ul
  10mM dNTP           0.5ul
  primer 1            1.0ul
  primer 2            1.0ul
  50:1 Taq:Vent       0.5ul  ALWAYS KEEP ENZYMES ON ICE!
  DNA                 1.0ul
  MQH20               18.5ul
    Total Volume: 25ul
  Run in thermocycler PCR program.

Gel Extraction

  Exracting DNA from gel purified samples - QIAquick gel extraction protocol

  Measure ng/ul concentration and A260/280 for the DNA sample by placing 1.5ul of DNA on the nanodrop.

Ligation

  Reaction mixture:
  vector DNA                  equal amounts of vector and insert are desired
  insert DNA                  compare concentrations C1V1 = C2V2
  10X NEB T4 ligase buffer    1.5ul
  T4 ligase                   1.0ul                   ALWAYS KEEP ENZYMES ON ICE!
  MQH2O                       as needed to bring up to total volume

  Incubate at 16 C for 30 min in thermocycler.
  May be stored at 4 C.

Transformation

  Thaw competent (XL1-B) cells on ice - ~15 min.
  Add 1.5ul DNA into each tube of cells.
  Cool on ice for 30 to 60 min.
  Heatshock cells at 42 C for 1 min.
  Cool on ice for 2 min.
  Add 900ul SOC media to each tube of cells.
  Incubate at 37 C, shaking, for 1 hr.
  Incubate LB + amp100 plates at 30 C to warm them.
  Plate 150ul of cells on LB + amp100 plates
  Incubate at 37 C overnight.

  Plating:
  Add 150ul of cells in SOC media to a sterile LB plate.
  Dip spreader in ethanol and flame to burn off the ethanol.
  Touch spreader to media, avoiding the cells on the plate, to cool it.
  Keeping spreader level, spin the plate so that the cells are evenly distributed around the plate.
  Let plate dry before inverting and incubating.

Colony PCR

  Reaction mixture:
  PCR 20 buffer                           2.5ul
  10mM dNTP                               0.5ul
  primer 1 (5mM)                          1.0ul
  primer 2 (5mM)                          1.0ul
  50:1 Taq:Vent                           0.5ul        ALWAYS KEEP ENZYMES ON ICE!
  DNA (cells: 1 colony in 25ul MQH2O)     1.0ul
  MQH2O                                  18.5ul

  Colony PCR thermocycler program:
  [96 C for 2 min
  96 C for 20 sec
  54 C for 20 sec
  68 C for 1 min 30 sec] (35X)
  4 C hold

Sequencing

  Reaction mixture:
  DNA                  1.0ul
  primer               1.0ul THAW IN 42 C WATERBATH
  Big Dye Mix          2.0ul KEEP ON ICE - THAW FOR 1 MIN ON BENCH BEFORE USE
  Sequencing buffer    2.0ul 
  Pellet Paint         1.0ul THAW IN 42 C WATERBATH, VORTEX 10 SEC
  MQH2O                3.0ul

  Sequencing thermocycler program:
  [96 C 10 sec
  50 C 20 sec
  60 C 2 min 30 sec] (30X)
  4 C hold

  Sequencing:
  Add 10ul 25mM EDTA to 1.5ml tube.
  Add 10ul sequencing reaction to tube.  Pipette to mix.
  Add 32ul 95% ETOH and vortex for 5 sec.
  Centrifuge for 3 min. to pellet.
  Aspirate liquid.  BE CAREFUL NOT TO SUCK UP YOUR PELLET!
  Add 200ul 70% ETOH and vortex for 5 sec.
  Centrifuge for 1 min.
  Aspirate liquid.  BE CAREFUL NOT TO SUCK UP YOUR PELLET!
  Dry with open lid in fume hood or in dark for 15 min.
  Take to MBSU for sequencing.

Annealing

  Resuspend DNA by centrifuging dry DNA tubes for 2 min. and adding 200ul Buffer EB.  
  Leave at room temperature for 10 min and then vortex for 30 sec.
  
  Reaction mixture:
  10X PCR 20 buffer   2.5ul
  DNA strand 1        5.0ul
  DNA strand 2        5.0ul
  MQH2O              12.5ul

  Anneal thermocycler program:
  90 C 1 sec
  90 C 10 sec
  -0.6 C / cycle (100X)
  4 C hold

Making a Glycerol Stock

  From fresh overnights, take 750ul of culture broth and add it to a stock tube.
  Add 750ul of 60% glycerol.
  Vortex for 30 seconds.
  Store at -80 C.

LacZ Assay

   Media:NINT_LacZAssay.pdf

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