Dissolving Bacmid

[sing09]

Hello everyone,
It is my first time preparing bacmids using the Bac-to-Bac protocol from Invitrogen. Everything went well until I added TE buffer to my samples, but they won't dissolve. I know I did not over dry the samples after washing with 70% ethanol. The protocol says to leave the samples in ice to dissolve(for 10 min. Yeah, right.), so I did not attempted to heat them up to 37C. I decided to leave them in 4C overnight. So, has anyone had this experience? Is it normal for bacmids to take so long to dissolve?
Many thanks in advance,
Sing09

[relaxin]

High mol. wt. DNA can be dissolved faster if you heat it at 37 ºC. The EDTA in TE buffer should inhibit any DNase activity. For genomic DNA, you can heat it up to 50 ºC.

[r.rosati]

Yes, it takes long for BACs to resuspend. As relaxin wrote, you can leave them for some hours at 37°C in TE buffer (or 1:10 diluted TE, that is 1 mM Tris, 0.1 mM EDTA, pH 8.0).
You might also need to resuspend them in a greater volume. Like genomic DNA, you only get it well in suspension when it's decently diluted.

[sing09]

Thank you both for your replies. I had left the bacmid samples in 4C over the weekend and still they haven't dissolved. I just placed them in 37C and hopefully they will dissolve. I think I should have enough TE buffer to dissolve. It was a miniprep from a 2 ml culture and I used 50 ul to dissolve.

Thanks a lot!
Sing09

[sing09]

Ok, guys. I am pretty much convinced that, to the contrary of what I thought, I probably over dried my bacmids. I already left my samples in 37C for hours and added 10 ul more TE buffer to bring the final volume to 60 ul. To make things worse, I have the precipitated DNA now stuck to the bottom of each tube because I had to do a short spin to get droplets down in order to add TE buffer. So, I've decided to increase the temperature to 55C (advice on a Qiagen protocol) and added 30 ul of endotoxin-free water. If this doesn't work, I don't know what to do anymore. Any additional ideas by any chance?

Many thanks to all,
Sing09

[relaxin]

That should work. Vortex it for a few second in the middle of incubation.

By the way, I believe it is endotoxin-free TE buffer (not water).

[sing09]

Hi guys, so here is the end of the bacmid saga: I did that prep once again and this time was careful to dissolve the plasmids in TE when they were still somewhat "wet" (1 ul droplet still on top). Once again, samples did not dissolve! So, I was suspicious of the kit and bought another one this time from Qiagen and the bacmids dissolved instantly! Cells have been transfected and now the protein is being expressed. Amazing, huh? After all, it is not hard to dissolve bacmids. I'm asking Invitrogen for my money back.

Thanks for your help!
Sing09

[relaxin]

I am not familiar with the Invitrogen kit. But I know when protein and DNA precipitate together, the pellet will be difficult to re-dissolve. The Invitrogen kit may not remove the protein completely.

[mchlbrmn]

Request refund of blood sweat and tears too. I'm sure they'll oblige promptly
[right :( ].

[sing09]

Yes, Relaxin, I think you're right. The Qiagen kit has extra steps for removing impurities. Good suggestion, mchlbrmn! You guys have a good day!
Sing09

[nyang]

Hello everyone, I have the same problem with my bacmid purified with Invitrogen's kit not dissolving. Sing09, which kit from Qiagen did you use?

Thanks!