7 replies to this topic

[jamessmith01]

I'm using the Active Motif ChIP-IT Express magnetic kit for ChIP experiments. The protocol says reverse cross-linking is done at 95ºC for 15 min or 65ºC for 2.5 hr, followed by Proteinase K treatment at 37ºC for 1 hr. However I've read most protocols elsewhere say anything between 4-6 hr or overnight at 65ºC for reverse cross-linking. Does anyone know if this protocol is okay, or should I just ignore it and go for a longer reverse cross-linking anyway? I had some weird PCR results and thought maybe incomplete reversal of cross-links could be an issue? Thanks.

Edited by jamessmith01, 08 March 2010 - 09:57 AM.

[annabellak]

We reverse X-link for either 15 mins at 95 degrees, or 5h at 65. I see no difference with either methods. Not sure about only 2.5h at 65  - that may not be enough. When I see weird results in my PCRs though it's usually due to the presence of inhibitors in the sample - diluting 1/10 helps.

[ezz]

annabellak, on Mar 9 2010, 12:48 AM, said:

We reverse X-link for either 15 mins at 95 degrees, or 5h at 65. I see no difference with either methods. Not sure about only 2.5h at 65  - that may not be enough. When I see weird results in my PCRs though it's usually due to the presence of inhibitors in the sample - diluting 1/10 helps.

Hi!

Can you tell me what proteinase inhibutors do you use and in which concentration?
I am using the ActiveMotif kit also and I do 5ul of PIC and PMSF for 1ml...

THX!

[jamessmith01]

ezz, on Mar 19 2010, 02:27 PM, said:

annabellak, on Mar 9 2010, 12:48 AM, said:

We reverse X-link for either 15 mins at 95 degrees, or 5h at 65. I see no difference with either methods. Not sure about only 2.5h at 65  - that may not be enough. When I see weird results in my PCRs though it's usually due to the presence of inhibitors in the sample - diluting 1/10 helps.

Hi!

Can you tell me what proteinase inhibutors do you use and in which concentration?
I am using the ActiveMotif kit also and I do 5ul of PIC and PMSF for 1ml...

THX!

I think annabellak was referring to inhibitors of PCR, such as SDS from the ChIP elution buffer.

[jamessmith01]

Just a quick update on this for anyone who may be doing a 15 min @ 95ºC reverse cross-linking:

We carried out reverse cross-linking of input (sheared chromatin) samples at 95ºC for 15 mins and 65ºC for 2, 4 and 6 hours. This was analysed by qPCR, and while there was no difference between the 65ºC samples, they all had Cts approximately one cycle lower than the 95ºC sample. Bearing in mind this was only the input sample, this effect might be different in ChIP DNA.

From now on I'll be doing at least a 2-hour treatment at 65ºC.

[KPDE]

jamessmith01, on Mar 24 2010, 08:50 AM, said:

Just a quick update on this for anyone who may be doing a 15 min @ 95ºC reverse cross-linking:

We carried out reverse cross-linking of input (sheared chromatin) samples at 95ºC for 15 mins and 65ºC for 2, 4 and 6 hours. This was analysed by qPCR, and while there was no difference between the 65ºC samples, they all had Cts approximately one cycle lower than the 95ºC sample. Bearing in mind this was only the input sample, this effect might be different in ChIP DNA.

From now on I'll be doing at least a 2-hour treatment at 65ºC.

The 95C for 15min is best done at a basic pH.  If your elution buffer is at pH 10 or above you should be ok.  Below this pH the DNA will become nicked and not be a as good a template for PCR (or may be degraded altogether).  In addition we've found that a little bit of chelating agent (1mM EDTA) can also help to preserve DNA at high temps.

[Mighty Mouse]

jamessmith01, on Mar 24 2010, 12:50 PM, said:

Just a quick update on this for anyone who may be doing a 15 min @ 95ºC reverse cross-linking:

We carried out reverse cross-linking of input (sheared chromatin) samples at 95ºC for 15 mins and 65ºC for 2, 4 and 6 hours. This was analysed by qPCR, and while there was no difference between the 65ºC samples, they all had Cts approximately one cycle lower than the 95ºC sample. Bearing in mind this was only the input sample, this effect might be different in ChIP DNA.

From now on I'll be doing at least a 2-hour treatment at 65ºC.

Just out of curiosity do you typically reverse cross-links just in your elution buffer? Or do you add NaCl? For what it's worth I typically elute and proteinase K treat at the same time (62C for 2hrs in a hyb oven to get some shaking; based on the Millipore MagnaChIP protocol) and then put at 95C for 10 min prior to purification. Seems to be working pretty well for me lately since I stopped using Millipore's elution buffer (which they won't tell me the recipe) and started using the more common 1% SDS + 0.1M NaHCO3.

MM

[jamessmith01]

Mighty Mouse, on Mar 26 2010, 12:42 PM, said:

jamessmith01, on Mar 24 2010, 12:50 PM, said:

Just a quick update on this for anyone who may be doing a 15 min @ 95ºC reverse cross-linking:

We carried out reverse cross-linking of input (sheared chromatin) samples at 95ºC for 15 mins and 65ºC for 2, 4 and 6 hours. This was analysed by qPCR, and while there was no difference between the 65ºC samples, they all had Cts approximately one cycle lower than the 95ºC sample. Bearing in mind this was only the input sample, this effect might be different in ChIP DNA.

From now on I'll be doing at least a 2-hour treatment at 65ºC.

Just out of curiosity do you typically reverse cross-links just in your elution buffer? Or do you add NaCl? For what it's worth I typically elute and proteinase K treat at the same time (62C for 2hrs in a hyb oven to get some shaking; based on the Millipore MagnaChIP protocol) and then put at 95C for 10 min prior to purification. Seems to be working pretty well for me lately since I stopped using Millipore's elution buffer (which they won't tell me the recipe) and started using the more common 1% SDS + 0.1M NaHCO3.

MM

Hi,

I follow the Active Motif protocol as below:

- Carry out washes.
- Add elution buffer ('AM2'), 15 min incubation.
- Add reverse X-linking buffer, remove supnt. from beads.
- Incubate 15 min @ 95ºC (or 2.5 hr @ 65ºC).
- Add proteinase K, incubate 1 hr @ 37ºC.
- Add proteinase K stop solution.

I'd assumed the reverse X-linking buffer contains NaCl, but maybe not?

Edited by jamessmith01, 30 March 2010 - 04:47 AM.