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Team:Alberta NINT/Protocols
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Lab Protocols
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Overnights
Inoculating LB culture tubes:
Add 5ul Amp50 or Amp100 to 200ml 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.
Digests
Reaction mix: DNA 5-15ul 10X NEB 2 buffer 10% of total volume 10X BSA 10% of total volume Enzyme 1 0.5ul 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.
