Hi all,
I have been successfully doing ATACseq until I started doing PCR amplification by myself. So far, I just gave tagmented DNA to core for PCR amplification and QC. These samples showed good distribution of DNA fragment size.
However, I decided to do everything by myself to reduce to cost.
The problem is the qPCR for determining additional cycles is not working.
Here are what I did.
After transposase reaction, DNA yields were 0.5~2ng/ul in total 20ul.
I ran 5 cycles of pre amplification as described in omni ATAC seq.
All primers were from Sigma in normal quality.
First I tried sybr mix and i5 i7 primer mix included in Kapa quantification kit, but didn't work.
Even with the primer I used for pre amplification, it didn't work.
There was amplification on standard DNA, so I don't think the reagent is defective(also it's new).
So I sticked to original omni-ATAC protocol which uses 0.65uM of primers, the same polymerase I used for the pre-amplification, and add Sybr green. Still, it didn't work.
I tried with 10x primer mix from the KAPA, but it didn't help.
So I just ran additional 10 cycles more for every samples, submitted them to the core for QC.
Surprisingly, half of samples were showing good enrichment of sizes around 150bp - 400bp, and the other half was only having large fragments ( > 2000 ).
it seems like although I have good tagmented DNA, somehow my primers are not constantly binding to adapter sequence of fragmented DNAs.
Has anyone had quality issue with primers?
Still, it doesn't explain why those good samples are not amplified in qPCR with both i5 i7 primer mix and the custom primers I used for pre amplification.
Could running qPCR on 384 wells be problem?
what are those huge size of DNA fragments after amplification seen from bioanalyzer data?
I would appreciate any comment from you guys. Thanks !!
I have been successfully doing ATACseq until I started doing PCR amplification by myself. So far, I just gave tagmented DNA to core for PCR amplification and QC. These samples showed good distribution of DNA fragment size.
However, I decided to do everything by myself to reduce to cost.
The problem is the qPCR for determining additional cycles is not working.
Here are what I did.
After transposase reaction, DNA yields were 0.5~2ng/ul in total 20ul.
I ran 5 cycles of pre amplification as described in omni ATAC seq.
All primers were from Sigma in normal quality.
First I tried sybr mix and i5 i7 primer mix included in Kapa quantification kit, but didn't work.
Even with the primer I used for pre amplification, it didn't work.
There was amplification on standard DNA, so I don't think the reagent is defective(also it's new).
So I sticked to original omni-ATAC protocol which uses 0.65uM of primers, the same polymerase I used for the pre-amplification, and add Sybr green. Still, it didn't work.
I tried with 10x primer mix from the KAPA, but it didn't help.
So I just ran additional 10 cycles more for every samples, submitted them to the core for QC.
Surprisingly, half of samples were showing good enrichment of sizes around 150bp - 400bp, and the other half was only having large fragments ( > 2000 ).
it seems like although I have good tagmented DNA, somehow my primers are not constantly binding to adapter sequence of fragmented DNAs.
Has anyone had quality issue with primers?
Still, it doesn't explain why those good samples are not amplified in qPCR with both i5 i7 primer mix and the custom primers I used for pre amplification.
Could running qPCR on 384 wells be problem?
what are those huge size of DNA fragments after amplification seen from bioanalyzer data?
I would appreciate any comment from you guys. Thanks !!
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