Zebrafish Care and Experimental Techniques: Midi-Prep of Plasmid DNA

Day 1: see "Bacterial Transformation by Heat Shock"

**Day 2: Starter Cultures and Inoculate into Large Bacteria Flasks**

NOTE: Starter culture is preferable, but not required; can immediately go to larger flasks if you want to

STARTER CULTURE:
1. Aliquot 2ml of the LB+Amp solution (0.02g Amp/200ml LB) into 14ml round bottom tubes
2. Using a yellow pipet tip, pick up an isolated large colony and drop the tip into the correct tube
3. Cap and place into incubator (37C) with shaker set to 200rpm for ~6 hours

Note: At this point, can refrigerate starter cultures until ready to inoculate and do midi-prep kit

INOCULATION:
1) Add Amp to LB broth at 100ug/ml (so, for 500ml of LB broth, add .05g Amp)
2) Add 100ml of LB+Amp into a 1L flask for every plasmid you are inoculating
3) Transfer 200ul of bacteria from starter culture into corresponding flask
4) Cover each 1L flask with aluminum foil
5) Incubate for 12-16 hrs in Dana Incubator (if necessary, use paper towels to prevent flasks from shaking too much)

**Day 3: Midi-Prep Kit**

NOTES:
-with new box, centrifuge any liquid in small tube before using it
-add LyseBlue and RNase A to P1 buffer first time kit is opened (all 110ul of each) and store in refrigerator
-store Buffers 1 and 3 in fridge

1) Pour bacteria from 1L flasks into large round, capped bacteria bottles (LB should be cloudy) and store on ice until ready to use
2) Centrifuge in supply room at 6000xg for 15 min (temp at 4C) (use JA-14 rotor); must turn on power before opening centrifuge
-use chart to convert g to rpm (note rotor #)
3) While centrifuging, take 95% ethanol and pour a bit into bacteria flasks and bacteria bottles; let sit for 10 minutes and then pour down the drain
4) Once done centrifuging (should see pellet at bottom), pour off excess liquid into a waste flask (one of the ones used for bacteria)
5) Add 4ml of Buffer P1 (in fridge) to each pellet
6) Vortex to re-suspend all the pellet into the liquid
7) Pour contents into LABELED 50 ml conical tubes
8) add 4ml of Buffer P2, shake vigorously, and let sit for 5 mins; soln should turn blue; this step is lyses the cells open
NOTE: THIS IS THE ONLY TIME SENSITIVE STEP. NO LONGER THAN 5 MINS.
9) While waiting, prepare filter cartridges by putting caps onto filters and labelling them; line filters up next to corresponding conical tubes
10) With 45 seconds left, open caps of conical tubes (should be gooey)--DO NOT MIX UP CAPS
11) After 5 mins, add 4ml of Buffer P3 and mix; blue should disappear; this step neutralizes the bacteria
12) Transfer liquid from conical tube into filter (with cap)and let it sit for 10 minutes
*Conical tubes now become waste containers*
13) While filter is sitting for 10 min, place column (QIAGEN-tip) onto conical tubes using blue holders and drain 4L of QBT buffer through column (equilibrating column)
14) After 10 minutes, plunge liquid from filter into LABELED column; DNA is binding to column
*14b) OPTIONAL: can run the lysate through the column 2x to ensure all the DNA has bound to column; if you do this, the conical tube that catches the liquid MUST be clean
15) Once lysate has gone through column, empty waste in conical tube (dump into waste flask)
16) Wash column 2x with 10ml of QC Buffer (empty waste container between the two washes)
17) Place filters into 14ml round bottom tubes
18) Elute DNA with 5ml of QF buffer into a clean 14 mL round bottom tube.

(Can stop here if necessary and proceed later--keep eluate on ice or @ 4C)

19) Precipitate DNA by adding 3.5ml of room temp isopropanol (IPOH, 2-propanol) and mix in 14ml round bottom tubes
20) Pour liquid into LABELED Beckman ultra clear tubes (will change with new tubes)
21) Centrifuge the Beckman tubes using ultracentrifuge in Dana for 30 min at 4C at >15000xg
-use swinging bucket rotor
22) Discard liquid from Beckman tubes; should see opaque pellets at the bottom of the tube (that's DNA!)
23) Place Beckman tubes upside down on paper towel
24) Place 1ml of 70% ethanol into each B. tube and pipet up and down GENTLY to get all of the DNA off the bottom
25) Put this 1ml soln into a new eppendorff tube (LABELED)
26) Microfuge epp. tubes for 10 min; small pellet of DNA on side should be visible
27) Pipet out excess liquid and lay on its side to let all excess ethanol to evaporate (~10 min); pellet should appear dry but still visible
28) Suspend DNA in 100-150ul of ddH2O
29) Flick to ensure that the DNA has dissolved into the water and store in fridge

NOTE: to clean waste beaker, add a splash of bleach, let sit for 10 minutes, pour down drain, and rinse thoroughly