From 2006.igem.org

Contents 1 Making Cells Competent 1.1 Media/Reagents 1.2 Pre 1.3 Competent Cells: Day 0 1.4 Competent Cells: Day 1 (optional) 1.5 Competent Cells: Day 2 2 Restrictions 2.1 Pre 2.2 Restricting 3 Ligation 3.1 Pre 3.2 Ligation 4 Transformation 4.1 Media/Reagents 4.2 Pre 4.3 Transformation

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:
   1. 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)).
   2. 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)
   3. If necessary (i.e. check NEB), add 1 μL 10X concentration BSA
   4. [x] μL of dH2O to make the total reaction volume in tube of 10 μL.
   5. 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.
2. (gingerly) flick tubes, and spin down in microcentrifuge for a second
3. Incubate at 37°C (in water bath or warm room) for 2-16 hrs. (3-4 hrs. optimal)
4. (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:

Stuff	Volume (uL)
insert	x
vector	y
dH2O	z
10X T4 ligase buffer	2
T4 ligase	0.5
Total	20

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)