Bac-to-Bac® Baculovirus Expression System (Invitrogen)
I have cloned a gene of interest into pFASTbac plasmid and this construct has been used to generate recombinant bacmid DNA. Blue and white colonies achieved following the bacterial transformation (DH10bac) and growth on antibiotic selection medium. To proceed to transfect the recombinant bacmid into insect cell lines I need to isolate the bacmid DNA. And here the problem arises. I am following the guideline given by Invitrogen. However, prior to transfection and in order to analyze the quality of the isolated bacmid, I run it on electrophoresis gel though (immediately after bacmid isolation). I expect to see the high molecular weight DNA BUT NOTHING IS THERE at all except the band representing RNA contamination.
I have also used some plasmid isolation procedures (mini/midi prep) but the result of electrophoresis is disappointing.
1. What could be the cause of the problem I encountered in Bacmid isolation? Why no DNA band could be traced on electrophoresis gel?
2. Is there a significant difference in tricks and points between the Bacmid and plasmid isolation procedures?
PS. I have considered the concerns regarding storage and handling
https://www.sciencedirect.com/topics/immunology-and-microbiology/bacmid
Hope this useful.
Bacmids are large and may not be efficiently isolated by standard kits.
There are specific kits for isolation of large DNA episomes such as BACs that may be good for you.
This is the protocol I was using back in the dark ages
B. Isolation of Bacmid:
1. Grow bacteria O.N in liquid medium containing 50µg/ml Kanamycin, 3µg/ml Gentamycin and 10µg/ml Tetracyclin.
2. Spin 1.5ml of culture @ 13000 RPM for 1min.
3. Resuspend pellet in 300µl of Sol1 (15mM Tris-HCl pH8.0, 10mM EDTA 100µg/ml RnaseA).
4. Add 300µl of Sol2 (0.2N NaOH, 1% SDS) mix gently and incubate @ RT for no more then 5min.
5. Slowly add 300µl of 3M Kac pH 5.5 while mixing.
6. Incubate 5-10min on ice.
7. Spin for 10min @ 13000 RPM. Meanwhile prepare a new tube with 800µl IsoPropanol.
8. Transfer sup to IsoPropanol containing tube (avoid white precipitant).
9. Mix by inversion and incubate 5-10min on ice.
At this stage its possible to leave sample O.N @ -200c
10. Spin 15min @ 13000 RPM.
11. Remove sup and wash with 70% EtOH.
12. Dry pellet and resuspend in 40µl H2O.
Dear Yoav
Thank you for the answer. I have used three plasmid isolation kits including Invitrogen (plasmid mini/midi prep), Thermo (plasmid mini prep) kits. Besides, I have already tried your recomended procedure which is also advised by Invitrogen and also a few other protocols. But all were in vain and the problem is still existing.
Could it be due to the very low copy number of the bacmid?? I mean in comparison to frequently used plasmids that are high copy number, bacmids are low in number and are larger in size. So, could it be solved by increasing the volume of the bacterial culture (maxi prep)?
I think the bacmid would exceed the maximum size that can be purified using standard kits so you're not likely to get much out of them.
You can try dedicated kits (we used the the Zymo ZR BAC DNA Miniprep Kit in the lab) but the procedure I mentioned have always worked for me.
It was about 15 years since I last did Baculovirus work so there may be some newer tricks out there I'm not familiar with.
We used to use ion excange maxi-prep kits for getting the bacmid DNA for bac-to-bac, and they worked, but I found that doing an old fashioned isopropanol precipitation was significantly more efficient. In some cases I now get more bacmid DNA from a 5 ml culture than I previously did with a 500 ml one, and I've not had any fail to transfect into the cells and make virus. I will admit that I've not checked them with electrophoresis though like you're doing, as it's not something that we do in our lab. Give me a while and I can put up the protocol I use if you want?
I use isopropanol precipitation to isolate the Bacmid. After lysing the cells, spin down and transfer the supernatant to pure isopropanol. Incubate on ice for 5 min and spin down. Your Bacmid is in the pellet. Wash it with 70% ethanol and then dissolve with water. This method never disappoints me
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