Post sonication RNAse & Prot K treatment - What are the bands? (Mar/10/2008 )
Thanx Davo for your answer,
How long do you sonicate your DNA and which settings do you use with the Branson-250?
-ChIPer-
QUOTE (Davo @ Apr 20 2008, 03:38 PM)
The reversing crosslinks is done with Prot K treatment - the histones will be degraded by the Proteinase K as they are proteins. As for the 10 mins at 95, that would be to inactivate the enzyme and maybe even be done to further undo the crosslinks. I got that step from the Upstate Magna ChIP method.
The 95C incubation is definitely important for reversal of the crosslinks. The majority of crosslinking reversal happens during this incubation. The rate of the reaction increases (as with any reaction) with higher temperature. Also, in this case, the rate of crosslink reversal is greatly enhanced with higher pH (a pH of 9.5 or higher).
-KPDE-
Why do many methods say to reverse crosslinks overnight at 55 deg? Why not skip straight to a much shorter 95 deg incubation?
I thought 62 deg incubation with Proteinase K was to keep the Proteinase K happy, but if you aren't using it, then are you saying a short incubation at 95 will have the same effect as overnight at 55? This could save some time 
Dave
-Davo-
QUOTE (Davo @ Apr 22 2008, 03:13 PM)
Why do many methods say to reverse crosslinks overnight at 55 deg? Why not skip straight to a much shorter 95 deg incubation?
I thought 62 deg incubation with Proteinase K was to keep the Proteinase K happy, but if you aren't using it, then are you saying a short incubation at 95 will have the same effect as overnight at 55? This could save some time
Dave
I thought 62 deg incubation with Proteinase K was to keep the Proteinase K happy, but if you aren't using it, then are you saying a short incubation at 95 will have the same effect as overnight at 55? This could save some time
Dave
I think some people have worried about incubating DNA at such a high temperature. At high temps, DNA can be nicked and amplification efficiency goes down. In the presence of chelex or a chelating agent + high pH, the DNA is presumably protected from damage. I agree with you. When we found out the short high temp incubation was as good as the long incubation at 65C, we wondered why we had been making it so difficult on ourselves before Fast ChIP.
-KPDE-
I think protease K treatment for 30 min and 95 C for 15 min are enough for reverse cross linking and the recoverd DNA does not need to purify through a DNA column if you use microplate well format. I got the kit and protocol from AmericanGenetech.com
QUOTE (Davo @ Apr 20 2008, 02:38 PM)
QUOTE (ChIPer @ Apr 20 2008, 12:41 AM)
Hi,
just a few questions. Isn't it necessery to do a reverse crosslink before the treatment with RNase and Prot K ? Why do you incubate your DNA for 10 min at 95°C. Won't you denaturate your DNA ? Of course it will renaturate again but why this step before you put in on the gel...?
I am doing sonication with a Branson-D250 and a microtip in an 1,5 ml Eppi volume about 500 ul? Whats the best position for the microtip in the tube...
Thanx a lot
just a few questions. Isn't it necessery to do a reverse crosslink before the treatment with RNase and Prot K ? Why do you incubate your DNA for 10 min at 95°C. Won't you denaturate your DNA ? Of course it will renaturate again but why this step before you put in on the gel...?
I am doing sonication with a Branson-D250 and a microtip in an 1,5 ml Eppi volume about 500 ul? Whats the best position for the microtip in the tube...
Thanx a lot
The reversing crosslinks is done with Prot K treatment - the histones will be degraded by the Proteinase K as they are proteins. As for the 10 mins at 95, that would be to inactivate the enzyme and maybe even be done to further undo the crosslinks. I got that step from the Upstate Magna ChIP method.
I use a Branson-250 sonifier. I had much better luck using 2ml tubes that have more of a flat bottom than the 1.5mls. I use 400ul but 500ul should be fine. I found with any less than 400ul and it was too easy to foam it all up = bad. I keep the tip anywhere between a few mm's from the bottom, and make sure it is at least a few mm's from the top. Too low and it won't work properly and too high and it will foam.
Good luck!
Dave
-johnd2008-
QUOTE (ChIPer @ Apr 21 2008)
Thanx Davo for your answer,
How long do you sonicate your DNA and which settings do you use with the Branson-250?
How long do you sonicate your DNA and which settings do you use with the Branson-250?
Sorry for the very late reply, I have only just re-read this thread in its entirety because someone posted a new comment.
With the Branson 250, I use an eight minute period of 30sec sonication, 30sec rest. I use a 60% duty cycle and output is turned to the lowest setting. When the tip pulses my sample, the output needle goes up to 20-40%. I keep my samples in -20 degree saturated NaCl solution and ice for the entire 8 minute period, and add a tiny amount of glass beads to the sample before sonicating.
Sorry it may be too late for you ChIPer, but it may be useful for others.
-Davo-