February 24, 2014

DAY 1 – Generation of a Recombinant Baculovirus in DH10-BAC

Generation of a Recombinant Baculovirus in DH10-BAC

Bacmid plates

Bacmid plates should be prepared in advance as follows:

Mix:

LB mix 25 g/l

Agar 15 g/l


February 25, 2014

DAY 2 – Generation of a Recombinant Baculovirus in DH10-BAC

Generation of a Recombinant Baculovirus in DH10-BAC

Transformation

Prepare 3 bacmid plates to have them warm by the time the cells are ready to be plated

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

Sit on ice for ~5 min

45 sec at 42 C

Spread the resulting transformation on 3 warm plates for each construct, so that one of the plate will have the right density of positive colonies:


Click to select a file on your computer, or drag here

Spread the resulting transformation on 3 warm plates for each construct, so that one of the plate will have the right density of positive colonies:

pKCC is the new plasmid needed to make this construct and not wait

pKCC
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10 µl on plate 1

lctm-image

lctm-image.jpg

100 µl on plate 3

Incubate at 37 C for 2 days

February 26, 2014

DAY 3 – Generation of a Recombinant Baculovirus in DH10-BAC

Generation of a Recombinant Baculovirus in DH10-BAC

Transformation results

Depending on the efficiency, the optimal colony density may be achieved in plate 1, 2, or 3

An optimal density is to have

Blue colonies 100-300


White colonies 5-20



Click to select a file on your computer, or drag the file here

Neuron spindles

Neuron spindles.jpg

nrm1272-i1

nrm1272-i1.jpg

Put in 3 ml LB medium with final concentrations:

Gentamicin 4 µg/ml

Kanamycin 25 µg/ml

Tetracycline 5 µg/ml

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

February 27, 2014

DAY 4 – Generation of a Recombinant Baculovirus in DH10-BAC

Generation of a Recombinant Baculovirus in DH10-BAC

Bacmid DNA preparation

Miniprep buffers

S1: 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 ug/mL RNase A (store at 4 C)

S2: 200 mM NaOH, 1% SDS (store at room temperature)

S3: 3.0 M potassium acetate pH 5.5 (store at 4 C)

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

Discard supernatant

Resuspend in 150 µl S1

Add 150 µl S2 and mix by inverting the tube 5 times

Incubate at R.T. for 5 minutes

Add 150 µl S3 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 excess 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