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FastChIP: is 200ul a large elution volume? - (Mar/18/2008 )

I have been reading over the FastChIP protocol and noticed that the final elution step is in 200ul of water. This is 4x what I use with the Upstate MagnaChIP kit and I'm wondering if the final DNA concentrations are similar or if I can expect a 4x drop in my qPCR results (which I can't afford).

thanks,
Kurt

-kman42-

QUOTE (kman42 @ Mar 18 2008, 04:55 PM)
I have been reading over the FastChIP protocol and noticed that the final elution step is in 200ul of water. This is 4x what I use with the Upstate MagnaChIP kit and I'm wondering if the final DNA concentrations are similar or if I can expect a 4x drop in my qPCR results (which I can't afford).

thanks,
Kurt


I can't speak for the yield of the Upstate kit but I know in my hands that the yield I got from Fast ChIP was about 10 fold better than what I got with the traditional assay (another colleague of mine saw a 2 to 3 fold improvement). In any case, you can always scale back the volume that you elute in. Others I've worked with have done this. Additionally, if you are working with small amounts of chromatin or with antibodies that pull down a small amount of chromatin and are only analyzing your results by PCR or QPCR, you might want to look into Matrix ChIP (Nucleic Acids Res. 2008 Feb;36(3):e17).

-KPDE-

QUOTE (KPDE @ Mar 20 2008, 03:17 AM)
QUOTE (kman42 @ Mar 18 2008, 04:55 PM)
I have been reading over the FastChIP protocol and noticed that the final elution step is in 200ul of water. This is 4x what I use with the Upstate MagnaChIP kit and I'm wondering if the final DNA concentrations are similar or if I can expect a 4x drop in my qPCR results (which I can't afford).

thanks,
Kurt


I can't speak for the yield of the Upstate kit but I know in my hands that the yield I got from Fast ChIP was about 10 fold better than what I got with the traditional assay (another colleague of mine saw a 2 to 3 fold improvement). In any case, you can always scale back the volume that you elute in. Others I've worked with have done this. Additionally, if you are working with small amounts of chromatin or with antibodies that pull down a small amount of chromatin and are only analyzing your results by PCR or QPCR, you might want to look into Matrix ChIP (Nucleic Acids Res. 2008 Feb;36(3):e17).






hi Joel

if i don't need that much water to perform PCR, do i need to do this? (Add 120 ml of water (MilliQ or NANOpure) to beads, vortex for 10 s, centrifuge contents down at 12,000g for 1 min of
4 1C, collect 120 ml of supernatant and pool with the previous supernatant. Mix before using.)

i need the ChIP product to have higher concentration of DNA, cause most of the time i can't get any band on gel after pcr .

thx a lot!

-yww-pride-

QUOTE (yww-pride @ Mar 20 2008, 07:24 AM)
QUOTE (KPDE @ Mar 20 2008, 03:17 AM)
QUOTE (kman42 @ Mar 18 2008, 04:55 PM)
I have been reading over the FastChIP protocol and noticed that the final elution step is in 200ul of water. This is 4x what I use with the Upstate MagnaChIP kit and I'm wondering if the final DNA concentrations are similar or if I can expect a 4x drop in my qPCR results (which I can't afford).

thanks,
Kurt


I can't speak for the yield of the Upstate kit but I know in my hands that the yield I got from Fast ChIP was about 10 fold better than what I got with the traditional assay (another colleague of mine saw a 2 to 3 fold improvement). In any case, you can always scale back the volume that you elute in. Others I've worked with have done this. Additionally, if you are working with small amounts of chromatin or with antibodies that pull down a small amount of chromatin and are only analyzing your results by PCR or QPCR, you might want to look into Matrix ChIP (Nucleic Acids Res. 2008 Feb;36(3):e17).






hi Joel

if i don't need that much water to perform PCR, do i need to do this? (Add 120 ml of water (MilliQ or NANOpure) to beads, vortex for 10 s, centrifuge contents down at 12,000g for 1 min of
4 1C, collect 120 ml of supernatant and pool with the previous supernatant. Mix before using.)

i need the ChIP product to have higher concentration of DNA, cause most of the time i can't get any band on gel after pcr .

thx a lot!


If you don't do the second elution I would suggest that you do one last wash using TE with a pH somewhere between 7.4 and 7.6. This should get rid of much of the remaining detergent in the buffer surrounding the beads (the dead volume which you can't aspirate off) which may be inhibitory to PCR at higher concentrations (i.e. when not diluted by the 120µl second elution).

Another thing you might try in order to improve your yield is use more chromatin and antibody without increasing the amount of beads. Or you could also try using less beads and blocking them with 5% BSA and 100µg/ml sheared salmon sperm DNA. The beads non-specifically bind up some of your chromatin and some of this cannot be eluted off. If you can decrease the beads' capacity to do this or increase the amount of chromatin and the specific binding capacity (your antibody) to overwhelm this non-specific retention, you may get a better yield.

Hope this helps.

Joel

-KPDE-

QUOTE (KPDE @ Mar 21 2008, 03:10 AM)
QUOTE (yww-pride @ Mar 20 2008, 07:24 AM)
QUOTE (KPDE @ Mar 20 2008, 03:17 AM)
QUOTE (kman42 @ Mar 18 2008, 04:55 PM)
I have been reading over the FastChIP protocol and noticed that the final elution step is in 200ul of water. This is 4x what I use with the Upstate MagnaChIP kit and I'm wondering if the final DNA concentrations are similar or if I can expect a 4x drop in my qPCR results (which I can't afford).

thanks,
Kurt


I can't speak for the yield of the Upstate kit but I know in my hands that the yield I got from Fast ChIP was about 10 fold better than what I got with the traditional assay (another colleague of mine saw a 2 to 3 fold improvement). In any case, you can always scale back the volume that you elute in. Others I've worked with have done this. Additionally, if you are working with small amounts of chromatin or with antibodies that pull down a small amount of chromatin and are only analyzing your results by PCR or QPCR, you might want to look into Matrix ChIP (Nucleic Acids Res. 2008 Feb;36(3):e17).






hi Joel

if i don't need that much water to perform PCR, do i need to do this? (Add 120 ml of water (MilliQ or NANOpure) to beads, vortex for 10 s, centrifuge contents down at 12,000g for 1 min of
4 1C, collect 120 ml of supernatant and pool with the previous supernatant. Mix before using.)

i need the ChIP product to have higher concentration of DNA, cause most of the time i can't get any band on gel after pcr .

thx a lot!


If you don't do the second elution I would suggest that you do one last wash using TE with a pH somewhere between 7.4 and 7.6. This should get rid of much of the remaining detergent in the buffer surrounding the beads (the dead volume which you can't aspirate off) which may be inhibitory to PCR at higher concentrations (i.e. when not diluted by the 120µl second elution).

Another thing you might try in order to improve your yield is use more chromatin and antibody without increasing the amount of beads. Or you could also try using less beads and blocking them with 5% BSA and 100µg/ml sheared salmon sperm DNA. The beads non-specifically bind up some of your chromatin and some of this cannot be eluted off. If you can decrease the beads' capacity to do this or increase the amount of chromatin and the specific binding capacity (your antibody) to overwhelm this non-specific retention, you may get a better yield.

Hope this helps.

Joel









THX! i'll try
best wishes closedeyes.gif

-yww-pride-