February 24, 2014
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
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:
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:
10 µl on plate 1
100 µl on plate 3
Incubate at 37 C for 2 days
February 26, 2014
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
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
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