November 6, 2009

DAY 1

Baculovirus recombination in bacmid

Transformation

Stock solutions:

Gentamicin stock 10 mg/ml in water

Kanamycin stock 50 mg/ml in water

Tetracycline stock 10 mg/ml in 70% ethanol

IPTG 200 mg/ml in water

Blue Gal 100 mg/ml in DMSO

I actually first used some older bacmid plates from Sept 2009 that did not yield any white colonies. The protocol below only talks about the good batch that was prepared by John last week. I should NEVER use the old plates again!

The transformation is performed as follows:

Make sure I have 3 bacmid plates

Mix 0.5 µg of pFastBac1-hETA and 10 µl DH10-BAC competent cells

Sit on ice for ~5 min

45 sec at 42 C

~1 min on ice

Add 130 µl LB

Incubate at 37 C for 2 hours

Spread the resulting transformation on 3 warm plates for each construct:

10 µl on plate 1

30 µl on plate 2

100 µl on plate 3

Incubate at 37 C for 2 days

November 7, 2009

DAY 2

Baculovirus recombination in bacmid

Transformation results

Colonies are at the best density in the plate with 10 µl

Blue colonies ~500

White colonies ~30

Miniculture for bacmid DNA preparation

Pick 2 white colonies on plate 1 and 2 on plate 2

The cultures are labeled, C151-B1,... C151-B4

Put in 3 ml LB medium with:

Gentamycine 4 µg/ml

Kanamycin 25 µg/ml

Tetracycline 5 µg/ml

Shaker overnight at 37 C, for ~20 hours, then store at 4 C

November 8, 2009

DAY 3

Baculovirus recombination in bacmid

Bacmid DNA preparation

See minicultures started yesterday = C151-B1,... C151-B4

Reagents = P1, P2, P3 from Qiagen

The Qiagen P3 comes with their Midi/Maxi kit. Do not use the N3 buffer from miniprep kit, it is not the same composition as P3 and it won't work.

Spin 1 ml of bacterial culture for 2 min at 13000 rpm at R.T.

discard supernatant

Resuspend in 150 µl P1

Add 150 µl P2 and mix by inverting the tube 5 times

Incubate at R.T. for 5 minutes

Add 150 µl P3 and mix by inverting the tube 10 times

Incubate at R.T. for 5 minutes

Centrifuge for 10 min at 13000 rpm at R.T.

Transfer supernatant to another clean microcentrifuge tube

Add 900 µl ethanol and mix by inverting 5-6 times

Centrifuge at 13000 rpm for 5 minutes at R.T.

Pour supernatant in the trash

Add 1 ml of 70% EtOH and remove by pouring in the trash

Briefly spin to bring down the residual ethanol (spin longer if the pellet was detached)

Carefully remove the remaining ethanol

Keep the tube open under the hood for 5-15 minutes to dry the pellet

Add 50 ul of of filtered water

Let it rest for 15 min at R.T. to fully resuspend

To avoid damaging and fragmenting the large bacmid molecules, do no pipet, instead just flick the tube a couple of times.

Use immediately for transfection

Store the remaining DNA at -20 C

DAY 1

Baculovirus strain from bacmid

Transfection of the bacmid DNA in insect cells

Using bacmid preparation C151-B1

Dilute 2 µl of bacmid DNA into 1 ml medium

Add 15 µl CellFectin and vortex briefly

Incubate at RT for 15 min

During that time, transfer to 50 ml Corning ~20 millions cells in log phase

Centrifuge the cells at 1000 rpm for 10 minutes

When everything is ready, gently tap the bottom of the 50 ml tube to resuspend the pelleted cells

Gently resuspend the cells in the transfection mix using a 1 ml serological pipette

Incubate in the shaker @ 28 C for ~5 hours

Add 19 ml medium and transfer to a small flask

Incubate in the shaker @ 28 C

The cells will then be counted every day and fed to maintain a cell density below 2 millions/ml, until all the cells show sign of virus infection and are dying, at which point the virus stock will be harvested

November 9, 2009

DAY 2

Baculovirus strain from bacmid

Cell counting and feeding

Cells density: ~0.2 million/ml, dividing

Sick cells: less than 10%

Add 10 ml medium -–> ~30 ml total

November 10, 2009

DAY 3

Baculovirus strain from bacmid

Cell counting

Cells density: ~0.5 million/ml, dividing

Sick cells: less than 10%

November 11, 2009

DAY 4

Baculovirus strain from bacmid

Cell counting

Cells density: ~0.5 million/ml, dividing

Sick cells: less than 10%

November 12, 2009

DAY 5

Baculovirus strain from bacmid

Cell counting and feeding

Cells density: ~1 million/ml, dividing

Sick cells: less than 10%

Add 10 ml medium, thus ~40 ml total

November 13, 2009

DAY 6

Baculovirus strain from bacmid

Cell counting

Cells density: ~1 million/ml, dividing

Sick cells ~10%

November 14, 2009

DAY 7

Baculovirus strain from bacmid

Cell counting

Cells density: ~1 million/ml

Sick cells ~40%

Staining with fluorescent antibody shows 20-50% cells expressing the receptor

Fluorescence Microscopy P1 - Day 5

Microscopy P1.png

P1 virus harvesting

Centrifuge 15 min at 20 C, 2000 rpm

Transfer sup to a new 50 ml Corning

The stock is labeled C151-1 P1

Store at 4 C

DAY 1

Baculovirus P2 production

Infection with P1

Start with 500 ml cells in log phase at 1 million cells / ml

Add 500 µl of one of the P1 virus stock C151-1 P1

Incubate in shaker at 27 C

The cells will then be counted every day and fed to maintain a cell density below 2 millions/ml, until all the cells show sign of virus infection and are dying, at which point the P2 virus stock will be harvested. This should take 2 to 4 days.

November 15, 2009

DAY 2

Baculovirus P2 production

Cell counting

Cells density: ~1 million/ml, dividing

Sick cells ~10%

November 16, 2009

DAY 3

Baculovirus P2 production

Cell counting

Cell count ~2 million cells/ml

All the cells look sick, not dying

Staining:

nothing on non-permeabilized cells

~10-30% good staining after permeabilization

Ready for harvesting

Harvesting after 2 days means we had a good titer on the P1 stock C151-1 P1 attachment

Fluorescence Microscopy P2 - Day 2

Microscopy P2.png

P2 virus harvesting

Infection time = 50 hours

Centrifuge 15 min at 4 C, 3000 rpm

Filter and transfer supernatant in sterile 250 ml bottles

Store this P2 virus stock at 4 C = C151-1 P2

Store pellets from 250 ml cells at -20 C to make membranes and check expression by Western Blot