Team:Alberta NINT/Protocols



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=Lab Protocols=

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|LacZ Assay PDF file]]