my problem with agilent Sureselect methy-seq kit

[ycding]

Hi, sorry for bothering you guys. I am using the agilent Sureselect methyl-seq target enrichment kit to generate library for illumina sequencer (high seq). after bisulfite treatment, I ran a QPCR to quantify how much DNA I got and only got about 20 ul 100 pM product, less than the expected concentration, 200 pM. I followed agilent protocol exactly. is any one using this kit and can give me some suggestion to improve the amount of product captured by this kit?

thank you so much in advance!!

ding

[ETHANol]

I haven't used this product but I would say the difference between 100 pM and 200 pM is pretty minor. It's just one extra PCR cycle. With that amount of DNA I would guess that you will only need about 10-12 cycles of PCR amplification which should not result in any significant amount of PCR duplicates.

But then again I've never used the kit.

[Agilent_Seq]

Hi, sorry for bothering you guys. I am using the agilent Sureselect methyl-seq target enrichment kit to generate library for illumina sequencer (high seq). after bisulfite treatment, I ran a QPCR to quantify how much DNA I got and only got about 20 ul 100 pM product, less than the expected concentration, 200 pM. I followed agilent protocol exactly. is any one using this kit and can give me some suggestion to improve the amount of product captured by this kit?

thank you so much in advance!!

ding

Hi Ding,

I’m a representative at Agilent and I would like to assist with your issue. Please contact [email protected] for a prompt response.

Thanks,
Agilent_Seq

[shawpa]

Hi, sorry for bothering you guys. I am using the agilent Sureselect methyl-seq target enrichment kit to generate library for illumina sequencer (high seq). after bisulfite treatment, I ran a QPCR to quantify how much DNA I got and only got about 20 ul 100 pM product, less than the expected concentration, 200 pM. I followed agilent protocol exactly. is any one using this kit and can give me some suggestion to improve the amount of product captured by this kit?

thank you so much in advance!!

ding

I was wondering if you finished your prep. I just finished and only 2 of 8 samples gave any output for me. How many cycles did you end up doing for the final pcr? I have only tried the suggested 4 along with the previous 9 from the other step.

[ycding]

I was wondering if you finished your prep. I just finished and only 2 of 8 samples gave any output for me. How many cycles did you end up doing for the final pcr? I have only tried the suggested 4 along with the previous 9 from the other step.

Hi thanks for your responde. I also did the 9 plus 4 cycles of PCR. I just got enought DNA for loadind to Illumina sequencer. a specialist in our bioinformatics core is analyzing my data.

out of 8 samples, what were the yields after bisulfite conversion for each sample ? do you get more than 200 pM?

[Taberlay]

Hi everyone,

I was wondering if there was any update on this issue.

1. Was the original issue resolved?
2. And/or, was there a compromise in the data quality/usability if less than 200pM was used?

I am currently facing this issue.

Thanks
Phillippa