Make Media for HEK293T Cells

500ml DMEM1. 5ml 100x Pyruvate

1. 5ml 100x Pen/Strep
2. 5ml 100x L-Glutamine
3. 50ml Fetal Bovine Serum

Thaw HEK293T Cells in 10cm Dish with 10 ml HEK Medium (One Vial into three 10cm Dish)

Split HEK293T Cells (Usually become confluent on Day 3) with 3ml Trypsin.

Use serum to block Trypsin (100ul for 1ml trypsin)

Split one full confluent 10cm dish into five 10cm plates (So Total 15 Plates).

Split all plates of HEK293T Cells (15 10 cm dish confluent plates)

Plate 15 million cells/T175 Flask with 20 ml Medium

Make sure all plasmids are ready on day of Transfection

pAd-DeltaF (Order from AddGene; Glycerol Stock-Cultivate at 30°C and do Maxi) 1. PHP.eB Capsid (Order from AddGene; Glycerol Stock-Cultivate at 37°C and do Maxi)

1. ITR-Gene of interest plasmid (i.e. GEARBOCS with gRNA for gene of interest)

In a sterile tube, dilute total plasmid DNA (ug) in 6ml Opti-MEM.

30 ug pAd-DeltaF1. 15 ug PHP.eB

1. 15 ug ITR-Gene of interest (pAAV2ITR-gfaABC1D-XXX)

In another sterile tube, dilute 1.33ml PEI (7.5mM) in 4.66ml Opti-MEM.

Incubate 10 minutes at RT.

Combine the two tubes, mix well and incubate 20 minutes at RT.

Add 2ml of DNA/PEI mixture to each plate of cells, mix well and return to incubator.

Change media (Usually 6-18 hours later)

Add 20ml fresh media and culture for 72hrs

Collect the cells and medium by scraping the cells off the dish with a cell scraper and transferring it to a 50-ml conical tube.

Rinse dishes with 2 ml of 1× PBS and transfer it to the same conical tube.

Harvest two dishes at a time into the same 50-ml tube

Centrifuge at 1300 rpm for 8 min in a tabletop centrifuge.

Aspirate the medium from the conical tube and repeat cell collection steps until cells are pelleted from all 6 dishes into one tube. The same tube can be used for pelleting cells from all the dishes.

Resuspend the final pellet from all 6 dishes in 4 ml of cell lysis buffer (In TC Room Fridge-50 ml Tube) (4 ml for one virus).

Prepare dry ice/ethanol bath during the last spin

Take dry ice in ice box and add 100% 190 proof ethanol to that to make dry ice/ethanol bath.

Freeze the pellet in the dry ice/ethanol bath and thaw in a 37 °C water bath three times (Almost 10-minute incubation each).

Freeze again in dry ice/ethanol bath and store at -80 °C. The pellet can be stored at −80 °C indefinitely and thawed at your convenience.

Hydrolyze nucleic acids

Thaw cell lysate in a 37 °C water bath1. Add 8ul of Benzonase to the 4 ml of thawed cell lysate at a final concentration of 50 U/ml (Benzonase stock conc. 25U/ul).

1. Incubate at 37 °C in a water bath for 30 min.
2. Centrifuge lysed cells at 4500 rcf for 30 min at 4 °C in a tabletop centrifuge

Prepare the iodixanol gradient during the Centrifugation.

The following volumes are for two gradients (or two viruses) using OptiSeal tubes. (Note: Always keep one extra. i.e if you have three viruses make gradient solutions for four)

Take four 50ml Falcon Tubes and label well each concentration of iodixanol

Add each component carefully to each tube and mix well by vortexing:

Iodixanol Gradient for two Viruses (Make one extra if you have 3 or more viruses)

A	B	C	D	E
	60% Iodixanol (OptiPrep Density Gradient-Sigma-D1556)# Don’t dilute	1 M NaCl/PBS- MK buffer	PBS-MK buffer	Phenol Red
15% Iodixanol	4.5mL	13.5mL	No	No
25% Iodixanol	5mL	No	7mL	30uL
40% Iodixanol	6.7mL	No	3.3mL	No
60% Iodixanol	10mL	No	No	45uL

Use a 10 or 12 ml syringe with an 18 G needle to add these solutions to each labelled centrifuge Optiseal tube in the order below.

(Notes before you add: Add very slowly along the sides of the tube and take care to avoid bubbles. The same needle can be used for loading all steps. Take exact volume otherwise 4ml virus cannot be accommodated over the top in last step)

i. 5 ml of 60% iodixanol (Bottom)

ii. 5 ml of 40% iodixanol

iii. 6 ml of 25% iodixanol

iv. 9 ml of 15% iodixanol step (Top)

Collect vector-containing supernatant from centrifuged tube using 10 or 12- ml syringe with 18G needle. The volume of the supernatant is approximately 4-4.5 ml.

Load the vector-containing supernatant over the iodixanol density gradient prepared before

Fill to the very top until the hinge of the tube. If you have space left over, top off the tube with cell lysis buffer but only until the hinge.

Close with the dark plug provided along with the Optiseal tube without anair bubble. If your tube does collapse and is hard to remove from the rotor, then drip acetone between the tube and rotor wall and extract with tweezers

Ultracentrifugation (67,000 rpm for 1 hour at 18°C)

Bring Beckman Ti70 rotor1. Put aerocap over the Optiseal Tubes

1. Keep tubes inside the rotor and press down
2. Tighten the lid and bring to the Beckman ultracentrifuge and keep inside
3. Close the lid and press vaccum (Green light blinks)
4. Press Speed-Press 67000-Press Enter
5. Press Temp- Press 18°C-Press Enter
6. Press Time- Press 1.00hr- Press Enter
7. After the vacuum green light is constant without blinking-Press Enter-Press START
8. Wait until it reaches the maximum speed before leaving centrifuge

Set up ring stand and clamp inside the hood to hold tube

Prepare bleach in bottle and keep inside the hood to discard tips and tubes

Once the centrifugation is done, bring rotor to TC room and open

Remove the aerocap from each tube using a pair of pair pliers

Remove black plug from each tube and discard into bleach bottle

Take out the Optiseal Tube with pliers by clipping the topside and place inside the hood

Spray down 70% alcohol to player, aerocap and inside rotor; and keep for 10 minute and wipe with tissue paper.

Keep ready the Millipore Amicon filter unit (UFC910008, 100K MWCO) with proper label

Keep the optiseal tube in the holder and tight well.

Lay a bunch of tissue paper down and keep the bleach bottle.

Puncture the tube on the side slightly below (3–5 mm) the 60–40% interface with an 18-gauge needle (bevel up) attached to a 10 ml syringe.

Slowly draw the 2-3 ml viral solution out from clear zone from each centrifuge tube by aspiration using the needle and put into the filter unit.

CRITICAL STEP: Avoid the proteinaceous material near the 40–25% interface. CRITICAL STEP : Avoid the proteinaceous material near the 40–25% interface.

Add 1ml of DPBS (#14190-144-Ca/MgCl2 Free) to filter unit and mix well using P1000

Spin 4000g for 10 minutes.

Repeat steps 43 and 44 five times.

In last step, concentrate up to 150-200ul

Collect virus from filter unit using P200 into a 1.6 ml Eppendorf tube

Aliquot the virus in 10ul amount to tubes and freeze in liquid N2

Store at –80 °C indefinitely