Dear friends,
I have a puzzle during BAC extraction.
The sample we extracted seems to be contaminated by RNA, although we digested it with RNase A and we cannot see the band for BAC in electrophoresis (0.8%).
But after we used RNase A to digest the sample, the band for BAC showed again.
The protocol we used is as follows:
RP24-97P16 BAC clone
1:1000 chloramphenicol incubate in 37 ℃ 14hr, 180 rpm
1) centrifuge 200ml bacterium solution in 12,000g 10min 4℃
2) abandon supernatant
3) resuspend pellet in12mL P1
(product of CWbiotech http://www.cwbiotech.com/, which I guess is similar to Qiagen
4) add 12mL P2, inverting 8 times, incubate in room temperature for 5 min
5) add 12mL P3, inverting 8 times, incubate in room temperature for 5 min
6) centrifuge 12000g 10 min 4℃
7) transfer supernatant to a new 50mL tube, digested with RNase A (Takara) 37℃ 30 min. Then add equal volume isopropanol
Incubate on ice for 30min
8) centrifuge 12000g 15min 4℃
9）add 5mL 70% EtoH to resuspend the pellet. Inverting pellet 5-10 times. And shake and beat the tube to let the pellet separate into small pieces.
10) 9000g 5 min 4℃
11）repeat 8~9
12) add 1mL 70% EtoH to resuspend the pellet. Transfer it to 1.5mL Eppendorf tube, inverting pellet 5-10 times. And shake and beat the tube to let the pellet separate into small pieces
13) 9000g 5min 4 ℃
14) dry it in room air
15) add 100uL ddH2O to dissolve sample
Run electrophoresis in 0.5X TBE.
The first picture is without being digested by RNase A.
The second one is after digestion.
The third one is a combination of undigested and digested sample.
Has anyone met such a problem before?
"But after we used RNase A to digest the sample, the band for BAC showed again."
What you are seeing is due to events that happen during the gel running. When the sample with the large amount of RNA runs through the gel, the massive amount of RNA "soaks up" all of the Ethidium Bromide in the lane as it moves (notice how the lanes above the large RNA band have a very low fluorescence background). The BAC DNA is still there in the lane, but you can't see it because there is no Ethidium Bromide staining of it. When you run the RNAsed sample, there is less RNA to soak up the Ethidium Bromide, so the BAC band "appears" again. If you post-stain the gel by soaking in Ethidium Bromide solution after the gel run, you should see the BAC band in both kinds of samples.
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