Creating Bacmid Jinqi Ren and Matt Barrett

Creating Bacmid Jinqi Ren and Matt Barrett

1/21/16

Supplies needed before starting:

Bacmid plates no more than 1 ½ months old. They contain

1)50 µg/ml Kanamycin,

2)7 µg /ml Genetamicin

3)10 µg/ml Tetracycline

4)100 µg/ml Bluo-Gal

5)40 µg/ml IPTG

Gene of interest in apFastBac vector.

DH10-Bac cells from invitrogen

Transformation:

1. Dilute your DNA and the supplied pUC19 DNA to 0.5 ng/µl. Add 1 ng (2 µl) of pFastBac vector to 25 µl DH10-Bac component cells, let sit on ice for 30 min. (I use 10ng/uL DNA and add 10ng, but the Invitrogen kit says use 0.5ng/uL and add 1ng.) Do the same for the pUC19 DNA.
2. Put each tube in a 42°C water bath for 45 sec and Put on ice for 2 min(optional). Place 300 µl of warm SOC media. Let shake for 4 hrs at 37°C.
3. During this time put two bacmid plates for each transformation into the 37°C incubator to warm up. Also put one ampicillin plate into the incubator.
4. After 4 hours, I add 20uL of transformed cellsonto one bacmid plate and 150uL to another. Each plate should provide you with plenty of colonies. Also the pUC19 transformed cell mixture I add 25uL onto an ampicillin plate. The Bacmid plates are already premade and contain.
5. Incubate samples on plates for 36-48hrs at 37°C. After 18hrs you should see colonies but you won’t be able to do a blue/white screening until 36-48hrs. The pUC19 plate should have colonies on the amp plate and there should be no colonies on the bacmid plates.

Pick white colonies for analysis, also pick one blue colony, for control.

Isolating Recombinant Bacmid DNA

1.For each virus to make, pick up 4 white colonies, and one blue colony. For each white colony that you pick with a pipet tip, touch it to another Bacmid plate,and then put the pipet tip into 4 ml LB containing 50 µg/ml Kanamycin, 7 µg /ml Genetamicin and 10 µg/ml Tetracycline. Grow both overnight at 37°C.

AntibioticStockFinal conc.If making 40 ml

Kanamycin50 mg/ml50 µg/ml40 µL

Gentamicin50 mg/ml7 µg /ml5.6 µL

Tetracycline5 mg/ml10 µg/ml80 µL

2. If any of the restreaked white colonies are blue the following day then pitch those liquid cultures, and continue on only with the liquid cultures that when restreaked are still white.

3.Centrifuge at 6,000rpm to pellet cells.

4.Remove the supernatant and resuspend the pellet with 0.3 ml Solution P1 (15 mMTris, pH 8.0, 10 mM EDTA, 100 µg/ml RNase A).

5.Add 0.3 ml Solution P2 (0.2 N NaOH, 1% SDS).

6.Slowly add 0.3 ml Solution N3 (3M potassium acetate, pH 5.5). mix gently. Place the sample on ice for 8 min.

7.Centrifuge for 10 min at 13,000rpm at 4°C

8.Gently transfer the supernatant to a microcentrifuge tube containing 0.8 ml of isopropanol. DO NOT transfer any white precipitate. Invert the tube a few times and place on ice for 8 min.

9.Centrifuge for 15 min at 13,000rpm at RT.

10.Carefully remove the supernatant, taking care not to disturb the pellet. Add 0.5 ml of 70% ethanol. Invert the tube several time to wash the pellet.

11.Centrifuge for 5 min at 13,000rpmat RT.

12.Remove the supernatant. Air dry the pellet for 5-10 min.

13.Dissolve the DNA in 25 µl of TE buffer. Pipet up and down to dissolve pellet only 5-6 times. If pellet is not dissolved, then the undissolved portion is white precipitate from step 7.

14.Store the DNA at 4°C.

Checking the bacmid by PCR

Check Bac-Bac Manual for full procedure. Quick outline below.

Use the M13 forward and reverse primers in a 50uL PCR reaction mixture.

For each sample, set up the following 50 μL PCR reaction in a 0.5-mL

microcentrifuge tube: 49uL master mix and 1uL of bacmid DNA.

Master mix
Chemical / Stock conc. / Final conc / Amount for one tube
PCR Buffer / 10X / 1X / 5 μL
dNTP Mix / 10 mM / 1 mM(0.25mM each) / 1.25μL
MgCl / 50 mM / 1 mM / 1μL
Primers / 10uM / 0.2μM / 1μL each
Sterile Water / N/A / N/A / 38.75
Pfu / 2 units/μL / 2 units/μL / 1μL

Recombinant bacmid DNA 1uL for most preps works fine. If you spec the bacmidDNA then add 100ng of DNA in sterile water.

Step / Time / Temp / Cycle number
Initial Denaturation / 2min / 93 / 1
Denaturation / 1min / 94 / 30
Annealing / 1min / 55 / 30
Extension / 2min/kb / 68 / 30
Final Extension / 2min/kb +2min / 68 / 1

.

3. Remove 5–10 μL from the reaction and analyze by agarose gel electrophoresis

PCR band size using both M13 forward and reverse primers is 2.5kd + size of gene of interest, using a pFASTBAC1 plasmid.