did u get any smear formation? have u run a DNA ladder marker? n for bands to be visible u need to add EtBr. How much did u add? u can try increasing its amount of bands are faint. How much DNA was loaded?

Hi Sanjaya =) I just tried this for the first time a few weeks ago. I isolated my DNA using phenol-chloroform extraction and then quantified and ran 5 ug of DNA on a 3% agarose gel (2% regular agarose plus 1% NuSieve agarose from Lonza) in 1X TAE buffer @ 80 volts. The first time I did the experiment, I used DNA extracted from mouse cerebellum. I didn't see any laddering. Although we see cell death in this tissue histologically, we now think it is not apoptosis and I think that is why we did not see any laddering. The second time, I used DNA extracted from mouse thymus that had been treated with a high dose of an alkylating agent, producing (histologically) a large amount of cell death. In this experiment I got laddering. The bands were a bit fuzzy so I think I need to play around with the gel percentage. 5 ug also was probably too much DNA so next time I will probably try 2.5 ug.

Are you sure your cells/tissues are undergoing apoptosis? If they are not, you may not see laddering. Sometimes late-stage necrosis cells will stain TUNEL-positive but I don't know if it also can give DNA laddering... What types of cells are you using? What treatment have they undergone?

Even "easy" methods in biology turn out to be difficult! The protocols seem so straightforward but you always have to do a lot of optimizing. =)

~catherine

yeah true.. brain tissue is always problematic for most of the techniques.. 89 volts seems to b too high unless you have a cooling arrangement in the apparatus. 50 volts or max of 70 volt if there is some emrgency... high voltage induces heating and causes hazing of bands due to melting of agarose.. 1.5 % gel is sufficent to see laddering.. if u want a protocol another than phenol chlorofm then gv me ur email id.. i hv heard there are sm protocols for the isolation of fragments only out of total DNA and then they can b run on gel for clearer laddering.. u can search for them

Thank you both Poonam and Catherine for passing on your ideas.

I have added the pic of the DNA ladder experiment with no ladder formation. When I took d pic, the marker is not seen clear though it could be seen a bit originally.

http://img89.imageshack.us/img89/1156/20101229dnaladderthatdi.jpg

I used Goldview TM an alternative for EtBr. The amount of DNA loaded was 10ul/well, with my concentration of DNA varying from 335 to 1395ng/ml. I did not try bringing them to equilibrium concentration before I carried this on. In terms of actual DNA added it thus becomes very low.

My sample are obtained from Oligodendroglial cell line following oxidative treatment and apoptosis is ascertained from flow cytometry too..I ran samples at 90V fr 70 mins.

I had not considered the melting of agarose at high voltages.... will keep in mind.

Well If you do have protocols for isolating the fragmented DNA in particular they might be more useful..my id is [email protected] you

Regards

Sanjaya

well sanjaya i do no have that protocol but i have heard about it , u can try searching on net.. well you should quantitate the DNA before leading at 280 nm and then load equal amount of DNA not equal quantity. it can be diiferent in all the well. Try using Etbr once, marker is not visible in your pic, try loading more of it. well your genomic DNA is visible nicely but fragments nor even smear is visible.. Have you run the gel to the completion? i see some dark things around centre of gel.. is that your loading dye?

Hello Poonam,

Thanks for the reply, well d marker was not good at d time, d next time i carried it out with another maker, it was ok but then DNA ladder was something lacking haha...yeah I was wondering in Western blot we load equal amounts in terms of protein...but then when I read on DNA ladder ..I found equal amounts in terms of volume..may be d step of bringing d concentration to equilibrium was missed hehe...By d way last time I quantified my DNA d concentration was very low. Under no circumstances will I be able to run 2.5ug/well as mentioned by Catherine.....what amount do u normally use. No actually I just ran it to d mid point and yeah the dark area near d center is d loading dye.

Will follow ur idea and try it later on thanks

Regards

Sanjaya

well ladder is always seen at the bottom of gel.. run the complete gel. only then u wl see fragments

Poonam, it depends on the dye and the percentage of the gel you are using if you will run the loading dye until the bottom of the gel. In an 1-1.5% agarose gel Orange G is running at roughly 50 bp whereas bromphenolblue is running at roughly 200-300 bp. Nevertheless, I agree that it should run longer than shown on the picture and the EthBr or any other staining dye should be stronger because actually you can see a small smear in the whole lanes. You can load as a negative control just normal genomic DNA from any source and as a positive control for the staining procedure sheared genomic DNA (via sonication).

Hello Nicole,

Thanks for your above comment, I had seen it before long- was sort of on vacation owing to the new yr holidays here, just started with the lab work again. As to the positive control preparation by sonification of genomic DNA, I found a protocol consisting of repeated cycles of sonification of cells for 10secs followed by cooling for 30 secs on ice bath... and in total of 10-15 such cycles. Then finally going doing the DNA extraction.

Is this the method u use or do you have any other methods for collecting positive control of fragmented DNA.

Thanks in advance...

Hi Sanjaya, welcome back! You should first lyse the cells in hypotonic buffer to remove the cytoplasmic fraction (usually for Chromatin precipitation with Formaldehyd crosslinking before, but you do not need it for your purposes). Afterwards solve the pellet in Ripa buffer to break the nucleus and then depending on the sonicator you can do in Ripa buffer something between 3-5 times (not too many) 10 sec pulse on a sonicator and 30-60 sec break in between with cooling the whole time in ice/ethanol bath. Finally, you perform a RNase and Proteinase digestion and purify the DNA either via Phenol/Chloroform and Ethanol precipitation OR via PCR purification kit (Qiagen for example). Do not do too many cycles of sonication because then your DNA will be too small. If you are not familiar with your sonicator, make several different samples: 1 cycle, 3 cycle and 5 cycle for example. And load in parallel part of the DNA purified without sonication. Good luck!

Thanks Nicole, u r too timely in answering to my question, thanks again.

I am using a local kit for the DNA extraction, so may be I will incorporate the sonification procedure once the cells are lyzed. I will follow ur idea and probably carry on the experiment soon.

Good luck with ur work too..

You are very welcome, Sanjaya! Good luck and let me know how it is going, please!

Hey Nicole,

I tried extracting the DNA using a kit and was trying to sonicate it using ice packs for intervals but as soon as the lysis buffer was kept on ice, it froze instantly, so could not use ice bath. Got to run it today or tomorrow. Hope this time i get something.

Will keep posted, cya

sanjaya

Hi Sanjaya, here is my protocol for extracting DNA to sonicate:

Resuspend the pellet in LYSIS BUFFER I containing proteases inhibitors and incubate on ice 20-60 min; discard supernatant (cytoplasmic fraction) and centrifuge the cells 1,800 rpm, 5 min, 4°C; resuspend the cell pellet in same volume LYSIS BUFFER II (RIPA buffer) + PI and incubate at 4°C 2 hrs-o/N, rotation. - LYSIS BUFFER I: 5 mM PIPES pH 8; 85 mM KCl; 0.5% NP40. LYSIS BUFFER II (RIPA buffer): 10 mM Tris-HCl pH 7.5; 150 mM NaCl; 1% NP40; 1% Sodium-Deoxycholat; 0.1% SDS; 1 mM EDTA.

For cooling use a bowl with ice and added enough cooled ethanol, that you will have an ice/ethanol bath. Ice packs might be too cold if they are still frozen and your buffer in your kit might be freezing even at around 4 degrees only. Good luck again, Nicole

Hello Everyone,

I am trying to performing the DNA ladder test as a measure for apoptosis. I isolated the DNA using a kit and also quantified it but I don't know how much DNA is needed for that assay and also the percentage of the gel on which it should be run

Hi there,

I too tried many times the ladder assay using genomic DNA extracted with the DNeasy kit (Qiagen) and loaded 3ug of DNA in 1.8% gel in Orange G buffer. I got the same result as Sanjaya: strong bands on top of the gel and no fragmentation. I did positive control incubating the cells with Staurosporine and still no laddering was observed...

Any suggestions?

Thank you!

Daniel

Check out this paper guys. Makes this DNA ladder assay pretty simple and definitely easy

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261451/

I tried few times the ladder assay using genomic DNA extracted with the DNeasy kit (Qiagen) and I used doxorubicin as a positive control, however I could not detect the laddering pattern of DNA. any help, did any one succeeded. is using qiagen method is suitable for DNA laddering detection. if yes why I couldnt detect it in the positive control