I have some problems with my sheared DNA now. I hope you can help me. Attached are two images for the same DNA gel. The images were taken at different times. One is at 1:00pm and the other is at 3:11pm.
It seems like I am getting some smeared DNA. But I do not know why there are blobs at the bottom of the band. I treated the samples with proteinase K, incubated them at 58 degree Celsius overnight. Then I did RNase A treatment at 37degree Celsius for 1 hour. After that, I ran an agarose gel at 53 voltage. Most sheared DNA bands on the web don't have blobs. Could you give me some suggestions about why that is?
Dear Xiuli Zhao,
The problem may be in your extraction procedure and not in Proteinase K Digestion, Incubation, or RNAse.
What you can do, just a suggestion, is that you can modify the DNA Extraction procedure. Or you can change the protocol.
May i know what procedure are you following for DNA Extraction.
Dr Imran Sabri
Your blobs look like RNA. Check if your RNase A is working effectively. But RNase usually used in Plasmid DNA isolation, not much in genomic DNA isolation. Maybe your sample is different.
I'm agreed with Pearl, you should check your RNase working or not. There is also possibility of your sample is not well digested (if you shear the DNA using enzymatic reaction)
HI, This is what I did before running a gel: To all the 10ul sheared samples, add ChIP elution buffer to a final volume of 50 uL
1. Add 1 uL Proteinase K and incubate at 58°C for 12 hours.
2. Add 1 uL of RNaseA (10 mg/mL, Sigma) and incubate for 60 minutes at 37°C.
3. Load 13 uL on a 1.3% agarose gel.
I will increase the amount of RNase A to see if the blobs are RNA. Thank you all for your reply.
Hi, I have another question. Since I tried many times and used all my cell lysis buffer and nuclei lysis buffer contained in millipore kit . I found many recipes for those buffers in web but I do not know which one would work well with millipore kit. Do you guys make your own lysis buffer? Do you have the experience to use them working with other reagents of millipore kit? Thanks a lot!
Another question: How do you shear you DNA? And how do you treat your DNA before and afterwards?
I don't think this is an RNA contamination because the small RNAs at the running front are missing. Besides that, RNAse A is a incredible robust enyzme which is really hard to kill.
By looking on the gel I would say that you break down your DNA not further than 300-500 nucleotides. Why this is, I don't know at the moment.
Hi Christian, I did sonication according with Millipore Kit protocol(17-408). Before sonication, I homogenized, added cell lysis buffer and nuclei lysis buffer. Sonication time were 20sec, 50sec, 80sec, 110sec for four samples, respectively. The weights of my samples were around 35mg. Sonication volume of each sample was 400ul. So I have high concentration samples for sonication. Now I feel my sonication is not enough. The blobs may be degraded ribosomal RNAs. Mouse rRNA sizes are around 1.9 Kb, 4.7Kb. I am going to increase the amount and time for my RNase treatment to double check。
I don't believe its RNA. An hour with RNAse A is more than sufficient to break down the RNA beyond recognition. And you should see much more small RNAs which are also missing.
Your blobs are getting stronger with longer sonication time, which may be due to more breakdown of gDNA and accumulation of small fragments.
Which sonicator do you use? We use one from Diagenode and here we sonicate for up to 15 minutes.
An easy test if this is RNA or not: Make one preparation with the RNAse, one without and treat them otherwise identical. Put them next to each other on the gel. I woud be really interested to see such a gel.
Xiuli, Why did you use ChIP elution buffer? If your experiment was ChIP assy, the blobs may be cross-linked gDNAs with proteins. Then, your PK may not work effectively. Check both your PK and RNase A.
Hi Christian, I tried what you said before. But I still got the blobs. I used 30 mg of Hippocampus but just added 1ul of RNase A. Maybe the RNase A is not enough.
I added 2ul RNase A now. Below is my gel. The blobs are not so bright as in previous gel(Jan 8). Obviously my soincation is not enough.
Interesting. Its been a while since I last did ChIP (luckily), but based on the last image, I would say that its probably your sonication that doesn't work properly. There is not much difference between your treated and untreated sample. And there seems to be very little DNA at all - can you use more tissue? When we did ChIP, we usually did this from cell culture cells and sometimes we used a number of 15cm dishes to get enough material. The other thing is the sonication, which of course depends on your sonicator. The type with a needle-shaped tip which is dipped into you solution require usually less sonication time than the waterbath-style sonicators.
Hi Pearl,
I do not know the exact function of ChIP elution buffer. I cannot get specific information about it from web. ChIP Elution buffer is used to elute the immune complexes. Do you use it in your protocol? Thanks!
Before you do ChIP you usually crosslink DNA and proteins, so you can fish the complex out of the soup using antibodies and some kind of beads. The ChIP elution resolves the DNA-Protein complexes and allows you to recover the DNA. If you don't elute, you cannot precipitate DNA in later steps.
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