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ChIP DNA quality


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#1 wgbiology

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Posted 01 April 2009 - 02:27 PM

I eluted my beads with 100 ul elution buffer (0.1M NaHCO3 and 1% SDS) twice and did decrosslinking at 65 degrees overnight, then added EDTA, Tris-HCL and Proteinase K, did digestion at 45 degrees for an hour, then purified the DNA with Qiagen PCR Purification Kit (28104). Briefly, I added 1000ul PBI buffer and 10ul NaOAC, after spinning, I eluted the ChIP DNA and INPUT DNA in 60ul and 100ul EB buffer, respectively. I took 2ul of DNA for realtime PCR, but it did not work at all for both ChIP and INPUT DNA except for the eIF4A control. I already tested some primers and some primers actually work well when I tested them. The weird thing is that when I diluted the ChIP DNA and INPUT DNA, realtime worked, but with low signals for the ChIP samples (Out of the range of standard curve). I tried several times with no significant improvement. It probably means that there are some inhibitors in the ChIP and INPUT DNA. Does anyone here have any good suggestions for my problems? Thanks a lot.
WGBIOLOGY

#2 KPDE

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Posted 02 April 2009 - 10:17 AM

I eluted my beads with 100 ul elution buffer (0.1M NaHCO3 and 1% SDS) twice and did decrosslinking at 65 degrees overnight, then added EDTA, Tris-HCL and Proteinase K, did digestion at 45 degrees for an hour, then purified the DNA with Qiagen PCR Purification Kit (28104). Briefly, I added 1000ul PBI buffer and 10ul NaOAC, after spinning, I eluted the ChIP DNA and INPUT DNA in 60ul and 100ul EB buffer, respectively. I took 2ul of DNA for realtime PCR, but it did not work at all for both ChIP and INPUT DNA except for the eIF4A control. I already tested some primers and some primers actually work well when I tested them. The weird thing is that when I diluted the ChIP DNA and INPUT DNA, realtime worked, but with low signals for the ChIP samples (Out of the range of standard curve). I tried several times with no significant improvement. It probably means that there are some inhibitors in the ChIP and INPUT DNA. Does anyone here have any good suggestions for my problems? Thanks a lot.
WGBIOLOGY


Just a thought, could you try an extra wash with the DNA cleanup kit. Also, check the handbook to see if there is anything in any of your buffers which is incompatible with (i.e. is not removed by) the kit you are using.

#3 wgbiology

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Posted 16 April 2009 - 02:02 PM

KPDE, Thanks a lot, I asked Qiagen technical service and they gave me the following advice, seems reasonable, but I have not tried it out.
BTW, I would love to try your Chelex fast ChIP method, do you mind sending me one copy? Thanks a lot.

The dye in PBI will not interfere with the downstream applications. However, the ChIP assays contain up to 1% SDS and our R&D did not test the compatibility of buffer PB or PBI with SDS. But there may be a risk of precipitation. Therefore you can dilute your assay to bring the SDS concentration down to 0.5 % and add 5 volumes of buffer PB. Alternatively, you could try using 1 vol PCR sample : 1 vol H2O : 5 vol PB, instead of 1 vol PCR sample : 5 vol PB. Also incubate the sample with Buffer PB/PBI for 30 minutes before applying it onto the column as in the paper below.

In addition, if needed, you can also add an extra wash with Buffer PE, or 80% ethanol to eliminate salt carry over.

Have a look at this Paper in Cell: Volume 115, pg 751-763, Dec 12, 2003. Here they have used QQ PCR to clean up assays. Combined sample with buffer PB and mixed well for 30 minutes prior to applying on column.


I eluted my beads with 100 ul elution buffer (0.1M NaHCO3 and 1% SDS) twice and did decrosslinking at 65 degrees overnight, then added EDTA, Tris-HCL and Proteinase K, did digestion at 45 degrees for an hour, then purified the DNA with Qiagen PCR Purification Kit (28104). Briefly, I added 1000ul PBI buffer and 10ul NaOAC, after spinning, I eluted the ChIP DNA and INPUT DNA in 60ul and 100ul EB buffer, respectively. I took 2ul of DNA for realtime PCR, but it did not work at all for both ChIP and INPUT DNA except for the eIF4A control. I already tested some primers and some primers actually work well when I tested them. The weird thing is that when I diluted the ChIP DNA and INPUT DNA, realtime worked, but with low signals for the ChIP samples (Out of the range of standard curve). I tried several times with no significant improvement. It probably means that there are some inhibitors in the ChIP and INPUT DNA. Does anyone here have any good suggestions for my problems? Thanks a lot.
WGBIOLOGY


Just a thought, could you try an extra wash with the DNA cleanup kit. Also, check the handbook to see if there is anything in any of your buffers which is incompatible with (i.e. is not removed by) the kit you are using.



#4 Clare

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Posted 17 April 2009 - 12:16 AM

I eluted my beads with 100 ul elution buffer (0.1M NaHCO3 and 1% SDS) twice and did decrosslinking at 65 degrees overnight, then added EDTA, Tris-HCL and Proteinase K, did digestion at 45 degrees for an hour, then purified the DNA with Qiagen PCR Purification Kit (28104). Briefly, I added 1000ul PBI buffer and 10ul NaOAC, after spinning, I eluted the ChIP DNA and INPUT DNA in 60ul and 100ul EB buffer, respectively. I took 2ul of DNA for realtime PCR, but it did not work at all for both ChIP and INPUT DNA except for the eIF4A control. I already tested some primers and some primers actually work well when I tested them. The weird thing is that when I diluted the ChIP DNA and INPUT DNA, realtime worked, but with low signals for the ChIP samples (Out of the range of standard curve). I tried several times with no significant improvement. It probably means that there are some inhibitors in the ChIP and INPUT DNA. Does anyone here have any good suggestions for my problems? Thanks a lot.
WGBIOLOGY


Hi there :lol:

We are using the same elution buffers and the same kit etc and our qPCRs work fine. Why are you adding NaOAc before purification? Could that interfere?

Our protocol after elution (total 200ul):

- add NaCl to a final of 0.3M and 1ul RNaseA (1mg/ml stock) - 67degC ON
- add 3ul PK (20mg/ml) at 45degC for 2 hours
- use the QIAGEN kit: We use buffer PB and not PBI as suggested by QIAGEN and follow the protocol. We elute in water (30ul for all samples) and not the Buffer EB.
- for qPCR we dilute the IP samples 1:4. This works beautifully :o We are using 6 million cells/IP.

Hope this helps :)
Have a great weekend!
Clare

#5 wgbiology

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Posted 17 April 2009 - 05:41 AM

Clare, Thanks a lot!
The reason for me to add NaOAC is that the pH of the eluted solution is not suitable for DNA binding to the column as shown by the color of PBI (the color is violet).
What do you think? Have you tried that? Do you always get high ratio of DNA recovery from the column?
By the way, I am using Arabidopsis plants for ChIP, it is quite different from animal cells.
Best regards,




I eluted my beads with 100 ul elution buffer (0.1M NaHCO3 and 1% SDS) twice and did decrosslinking at 65 degrees overnight, then added EDTA, Tris-HCL and Proteinase K, did digestion at 45 degrees for an hour, then purified the DNA with Qiagen PCR Purification Kit (28104). Briefly, I added 1000ul PBI buffer and 10ul NaOAC, after spinning, I eluted the ChIP DNA and INPUT DNA in 60ul and 100ul EB buffer, respectively. I took 2ul of DNA for realtime PCR, but it did not work at all for both ChIP and INPUT DNA except for the eIF4A control. I already tested some primers and some primers actually work well when I tested them. The weird thing is that when I diluted the ChIP DNA and INPUT DNA, realtime worked, but with low signals for the ChIP samples (Out of the range of standard curve). I tried several times with no significant improvement. It probably means that there are some inhibitors in the ChIP and INPUT DNA. Does anyone here have any good suggestions for my problems? Thanks a lot.
WGBIOLOGY


Hi there :blink:

We are using the same elution buffers and the same kit etc and our qPCRs work fine. Why are you adding NaOAc before purification? Could that interfere?

Our protocol after elution (total 200ul):

- add NaCl to a final of 0.3M and 1ul RNaseA (1mg/ml stock) - 67degC ON
- add 3ul PK (20mg/ml) at 45degC for 2 hours
- use the QIAGEN kit: We use buffer PB and not PBI as suggested by QIAGEN and follow the protocol. We elute in water (30ul for all samples) and not the Buffer EB.
- for qPCR we dilute the IP samples 1:4. This works beautifully ;) We are using 6 million cells/IP.

Hope this helps :)
Have a great weekend!
Clare



#6 Clare

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Posted 17 April 2009 - 07:24 AM

aahh I see...
Have you tried Buffer PB? Or a different solution to change the pH? I think something funky is happening during your purification which affects your qPCR.
I have never had to adjust the pH (we are using human cells).
Also, I never check how much DNA I have post-ChIP. As we are using such low amounts of starting material, I don't want to waste any trying to quantify it.
Clare

[quote name='wgbiology' date='Apr 17 2009, 02:41 PM' post='22084']
Clare, Thanks a lot!
The reason for me to add NaOAC is that the pH of the eluted solution is not suitable for DNA binding to the column as shown by the color of PBI (the color is violet).
What do you think? Have you tried that? Do you always get high ratio of DNA recovery from the column?
By the way, I am using Arabidopsis plants for ChIP, it is quite different from animal cells.
Best regards,




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