Editing Protein Production using Baculovirus (section)

== Cloning<br>  ==

<br>1.Clone target gene into pFastbac1 or pFastBac Dual<br>If His tag is required, add 6XHis tag to N-terminus. 

GGATCC ATG TCG TAC TAC CAT CAC CAT CAC CAc CAt atg 

If TEV site is needed, add the following sequence: <br>GGATCC ATG TCG TAC TAC CAT CAC CAT CAC CAT CAC GAT TAC GAT ATC CCA ACG ACC GAA AAC CTG TAT TTT CAG GGC catatg 

<br>2. Transform DH5alpha, Top10 or NEB Turbo, grow on LB/amp plate 

3. Make plasmid and verify plasmid by restriction digestion, or sequencing 

4. Make grlycerol stock. 

Transform DH10Bac 

1. Grow DH10Bac in 50 ug/ml kanamycin, 10 ug/ml tetracycline to log phase<br>2. Use CaCl2 protocol to make competent cells 

<br>3. Make 3 LB agar plates for each construct, <br>LB agar plate:<br>50 ug/ml kanamycin, 7 ug/ml gentamicin, 10 ug/ml tetracycline, 100 ug/ml Bluo-gal, and 40 ug/ml IPTG 

4. Transform DH10Bac using recombinant plasmid. 

5. Spin down cells, resubspend in remaining ~ 100 µl SOC medium, spread 15 µl to one plate, and 85 µl to another plate. 

6. Incubate plates for 48 hours at 37 °C 

7. Pick 10 most isolated large white colonies, and restreak them on fresh LB agar plates containing<br>LB agar plate:<br>50 ug/ml kanamycin, 7 ug/ml gentamicin, 10 ug/ml tetracycline, 100 ug/ml Bluo-gal, and 40 ug/ml IPTG. Incubate the plates overnight at 37 °C. 

8. Pick 1 isolated large white colony from each parental colony, inocµlate a liquid cµlture containing 50 ug/ml kanamycin, 7 ug/ml gentamicin, 10 ug/ml tetracycline. 

9. Make miniprep. Run gel to see if it is a big DNA. 

10. Store bacmid DNA at –20°C. 

11. Make glycerol stock and store at –80°C.