Invitrogen Bac to Bac Expression System
Invitrogen Bac to Bac Expression System
Invitrogen Bac to Bac Expression System
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Troubleshooting, continued<br />
Transfecting Insect Cells, continued<br />
Problem Reason Solution<br />
Low yield of virus,<br />
continued<br />
<strong>Bac</strong>mid DNA is not pure (i.e.<br />
contains recombinant bacmid<br />
and empty bacmid)<br />
• Screen other DH10<strong>Bac</strong> <br />
transformants and choose one that<br />
contains only recombinant bacmid.<br />
• Perform plaque purification <strong>to</strong><br />
isolate recombinant baculovirus.<br />
Expressing Your<br />
Protein<br />
The table below lists some potential problems and possible solutions that may<br />
help you troubleshoot your expression experiments.<br />
Problem Reason Solution<br />
Low protein yield Viral s<strong>to</strong>ck contains a mixture of<br />
recombinant and nonrecombinant<br />
Perform plaque purification <strong>to</strong> isolate<br />
recombinant baculovirus.<br />
baculovirus<br />
<strong>Bac</strong>ulovirus not recombinant • Verify transposition by PCR<br />
analysis of bacmid DNA using the<br />
M13 forward (-40) and M13 reverse<br />
primers.<br />
• Re-transfect insect cells with new<br />
recombinant bacmid DNA.<br />
Used <strong>to</strong>o low or <strong>to</strong>o high viral<br />
titer<br />
Time of cell harvest not optimal<br />
Cell growth conditions and<br />
medium not optimal<br />
Cell line not optimal<br />
Optimize infection conditions by<br />
varying the MOI.<br />
Perform a time course of expression <strong>to</strong><br />
determine the optimal time <strong>to</strong> obtain<br />
maximal protein expression.<br />
• Optimize culture conditions based<br />
on the size of your culture vessel<br />
and expression conditions.<br />
• Culture cells in Sf-900 II SFM for<br />
optimal cell growth and protein<br />
expression.<br />
Try other insect cell lines.<br />
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