Penn State University 2006:PSUprotocols

=Making Cells Competent= Time: O’N then 3 hr next day

''Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 "Trans. Using CaCl2" ''

Makes 64x50μL aliquots

Media/Reagents

 * LB
 * Cells (from plate)
 * CaCl2 Solution
 * Dry Ice (2nd floor Althouse or S. Frear)

Pre

 * Prepare CaCl2 solution1
 * Autoclave centrifuge bottles/tubes and chill on ice
 * Before centrifuge steps, make sure centrifuge is on and at 0°C

Competent Cells: Day 0

 * 1) Streak cells on LB plate
 * 2) Grow overnight at 37°C

Competent Cells: Day 1 (optional)

 * 1) Inoculate overnight starter culture (2 mL) w/ colony from plate

Competent Cells: Day 2

 * 1) Inoculate 80 mL2 with appropriate amount of O’N to obtain OD6000~0.1. Place cells on ice at OD600=0.375 (do not allow growth past an OD of 0.4, as this decreases subsequent transformation efficiency).  Typically this will take 40-60 min (check every 5 min when OD is getting near 0.375).
 * 2) Ice for 10 min.
 * 3) Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
 * 4) Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl2 buffer.
 * 5) Spin 1100g/5 min/0°C.
 * 6) Gently resuspend pellet in 8 mL ice-cold CaCl2. Let stand on ice for 30 min.
 * 7) Spin 1100g/5 min/0°C. Decant supernatant & resuspend well in 1.6 mL ice-cold CaCl2.  Cells may stand on ice for 12-24 hrs to increase competency.
 * 8) Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs. Immerse immediately in crushed dry ice.
 * 9) Transfer to box(es) and store in -80°C freezer (downstairs on 2nd floor).

1CaCl2(per L)
 * 20 g bactotriptone
 * 5 g bacto-yeast extract
 * 0.5 g NaCl
 * 0.19 g KCl
 * Adjust to pH 7.0 w/ NaOH
 * Autoclave
 * Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)

2Procedure can be scaled up or down as necessary

=Restrictions= Time: 20 min

Reference: NEB.com

Pre

 * (optional) Determine plasmid concentration via A260 measurements or by comparing intensities of bands on gel with those of the ladder DNA (whose masses are known).

Restricting

 * 1) To an eppendorf tube, add:
 * 2) Appropriate volume of plasmid for a total of approx. 700 ng DNA (usually, if plasmid prep is good, this will be 2 μL (i.e. ~350 ng/ μL)).
 * 3) 1 μL of appropriate 10X concentration buffer (to determine correct buffer check compatibility of restriction buffers using NEB catalogue or going online to NEB.com)
 * 4) If necessary (i.e. check NEB), add 1 μL 10X concentration BSA
 * 5) [x] μL of dH2O to make the total reaction volume in tube of 10 μL.
 * 6) Chosen restriction enzymes. They are at a high enough concentration that 0.5 μL of each is more than sufficient for a restriction digest.  ALWAYS ADD THESE LAST (and work quickly), in order to minimize time out of the freezer.  Keep these enzymes in their low-temp blue carrying case when out of the freezer.
 * 7) (gingerly) flick tubes, and spin down in microcentrifuge for a second
 * 8) Incubate at 37°C (in water bath or warm room) for 2-16 hrs. (3-4 hrs. optimal)
 * 9) (under review)...treat any vectors w/0.2 μL CIP and incubate for 30 min at 37°C with rest of restrictions

=Ligation= Time: 15 min

''Reference: NEB T4 ligase technical bulletin http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0202.pdf''

Pre

 * Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio.

Ligation

 * 1) for 20 μL reaction volume, to eppendorf add:

where x is usually 5-15 uL, y=[1,5] uL.

Also, always remember to do a negative control, which will be like the reaction above, but w/ no insert. Your transformation plates will then be an measure of any vector re-ligation/uncut background.

2. For overnight ligation, add ice to cold water in styrofoam container to bring temp to 12-14ºC. Incubate overnight (15 hr.). Alternatively, for quicker ligation, ligate at RT for 1 hr. Proceed directly to transformation.

=Transformation= Time: 2.25 hr

Media/Reagents

 * Competent cells
 * SOC1
 * Antibiotic LB plates

Pre

 * Chill ligated DNA on ice
 * Thaw competent cells ((found in eppendorfs in box on second-shelf down of -80ºC freezer) on ice for ~5 min.

Transformation

 * 1) Add 3 μL ligation mixture to cells in bottom of tube
 * 2) Incubate on ice for 30 min
 * 3) Heat shock in 42ºC water bath for 1 min (warm SOC media at same time).
 * 4) Incubate on ice for 2 min
 * 5) Add 500 μL warm SOC media
 * 6) Shake in 37ºC room for 1 hr
 * 7) Spread 500 μL on LB/antibiotic plates using rotator and glass spreading wand.
 * 8) Incubate plates in 37ºC room overnight.

1To make SOC, add 200 μL 500 mM filter-sterilized glucose to 5 mL SOB (20 mM final glucose concentration)