Calcium phosphate transfection protocol

1. Grow 293 cells in DMEM + 10% FCS + Pen/Strep at 37°C and 5% CO2 (split 1/10 every 2 days)

2. Split cells to about 50% confluency at least 2 hrs prior to transfection.

Cells must be low passage & do not allow them to be over-confluent (max 60%).

Incubate plates at 37oC overnight. If cell is 50~70% confluency after plating, you can do transfection in the morning.

Overgrown cells should not be used for Ca transfection.

3. Thaw 2X HBS at room temperature. Do not refreeze HBS after use.

4. Prepare CaCl2/HBS/DNA nanoparticles (10 cm dish)

aliquot 0.5 ml 2X HBS into a sterile 1.5 ml microfuge tube

In a separate tube:

Add X ÎŒl of ddH20 to the tube (final vol is 0.5 ml)

Add 15 ÎŒg of lentivirus vector with GOI

Add 15 ÎŒg of 3:1 pGag-Pol/pVSVG

Add 50 ÎŒl of 2.5 M CaCl2

Add 500 ÎŒl of 2x HBSS to CaCl2/DNA mix, mixing gently during the addition.

Incubate at RT for 20 min.

Add mixture to cells by dropping slowly and evenly into medium, swirl plate gently.

Incubate at 37°C/5% CO2 for overnight

Note: 2x 10cm plates (when concentrated down to 100 ÎŒl), should give titres around 10⁹-10¹⁰

For 150 mm Dish

30 ug DNA

160 ul 2 M CaCl2

1.3 ml 2XHBS

5. Remove media and add 10 ml fresh medium

2X HBS

8g NaCl

0.2 g Na2HPO4-7H2O (0.14 g if using dihydrate; [phosphate] must be 1.5 mM)

6.5 g HEPES

pH to 7.0 and bring up to 500 ml with distilled water. Re-pH at this point because it can change. Aliquot and store at 20°C.

or

2X HBS 500 ml

HEPES (Acid) 5g

NaCl 8g

Dextrose 1g

KCl 3.7g

Na2HPO4(7H2O) 10ml of Na2HPO4(7H2O) *Stock Solution (see below)

- Add ultrapure H2O up to ca. 450ml

- Adjust pH to exactly 7.1

- Add ultrapure H2O up to 1L

- Filter through a 0.2u sterile filter

Aliquot into sterile tubes and keep frozen at 20 oC. Keep one working tube at 4oC.

Note: once thawed, do not re-freeze

• Na2HPO4(7H2O) Stock Solution = 0.94 g of Na2HPO4(7H2O) in 50ml ultrapure H2O (Aldrich

Cat#32,007-2).

2.5M CaCl2 100ml

CaCl2 27.75g anhydrous CaCl2

Or CaCl2 36.75g CaCl2(2H2O)

Add water to 100 ml, filter sterilize and store at aliquot into 1.5 ml tubes, store at 4 °C or - 20 oC.

This table is from another protocol, for reference only, do not use--

Format	Relative  Surface Area	Culture media	Transf. Media	2X HBS	2.5M CaCl2	Total DNA	GFP
24 well	0.25X	500µl	280µl	18µl	1.1µl	1.1µg	3.6-9.0ng
12 well	0.5X	1ml	570µl	36µl	2.1µl	2.1µg	7.1-18ng
6 well	1.2X	2ml	1.4ml	86µl	5.1µl	5.1µg	17-43ng
35mm	1.0X	2ml	1.15ml	71µl	4.3µl	4.3µg	14-36ng
60mm	2.6X	5ml	3ml	186µl	11µl	11µg	37-93µg
10cm	7X	10ml	8ml	500µl	30µl	30µg	100-250ng
T25	3X	5ml	3.4ml	214µl	13µl	13µg	43-107ng
T75	9X	12ml	10ml	643µl	39µl	39µg	128-321
T150	18X	24ml	20.5ml	1.3ml	77µl	77µg	258-643

Day 3) Check transfections:

- Cells should be reaching full confluency

- Check fluorescence (if plasmid has a fluorescent tag)

- Transfection efficiency should be around 90%

Note: when you convert from “bright light” to “fluorescence” you should be able to see that approximately 90% of cells are positive for fluorescence.

- Remove media

- Add 10 ml of fresh DMEM + 2% FBS to each dish - Incubate overnight at 37°C/5% CO2

Day 4)

Viral Harvest # 1:

- Collect & pool supernatants (if you have multiple plates per vector)

- Spin at 1500 rpm for 5 min to pellet any cells/cellular debris

- Collect supernantant with a syringe & filter through a 0.45 ÎŒm syringe filter into a “NEW” pre-labelled falcon tube

- Cover falcon tube containing filtered virus supernatant with parafilm & place into sealable bag (double containment)

- Store at 4oC overnight in a cardboard box

- Add 10 ml of fresh DMEM + 2% FBS to each dish

- Transfer to 37°C/10% CO2 incubator - Incubate overnight

Day 5) Viral Harvest # 2:

- Collect & pool supernatants (if you have multiple plates per vector)

- Spin at 1500 rpm for 5 min to pellet any cells/cellular debris

- Collect supernantant with a syringe

- Pass supernatant through a 0.45 ÎŒm syringe filter into the falcon tube containing your filtered “Viral Harvest # 1” supernatant from the previous day

Note: The cleared supernatant may be used to directly transduce cells & titre will be relatively low (~106 viral particles/ml)

Day 5) Virus concentration (for in vitro grade stock):

- Centrifuge the supernatant using conical tubes and a Beckman ultracentrifuge with a SW41 swing bucket rotor (makes it easier to detect the viral pellet)

- spin at 24,000 rpm for 1 hr at 4°C

- carefully poor off supernatant without disrupting viral pellet

- add last remaining supernatant & spin again (do not use these tubes more than 3 times)

- carefully poor off supernatant

- allow remaining supernatant to drain off by resting tubes on paper towel

- aspirate all remaining fluid

- Resuspend viral pellet in 100 ÎŒl of 1x HBSS (no frothing)

- Rinse tubes with 50 ÎŒl of 1x HBSS & pool the samples (total volume per tube = 150 ÎŒl)

- Transfer to ‘screw-top’ microcentrifuge tubes or eppendorf tubes covered with parafilm

- Vortex at low speed for 15-30 min

- Spin 10 seconds using a table top microcentrifuge

- Collect supernatant (140 ÎŒl).

Note:

Use 40 ÎŒl (max) to determine viral titres by FACS or p24 ELISA.

Customers get 100 ÎŒl Short term storage (< 1 mth) at -20°C; Long term storage (> 1 mth) & at -70°C.