Methylated DNA Quantification Kit (Fluorometric) (ab117129)

Having reviewed this case with the lab, I would like to offer some suggestions to help optimize the results from ab117129.

It appears that the signal may be saturated either due to

1) insufficient washing or
2) too much time between adding Fluoro-Development Solution and reading the plate.

We have the following recommendations to improve the assay results:

1) add an additional wash at each wash step making sure to remove all residual wash solution from the wells at each step and especially before the signal detection step using a multi-channel pipette; and
2) reduce the color development time after adding the Fluoro-Development Solution.

The color development is continuous and cannot be stopped therefore after some time the signal will reach plateau for all of the wells. In general, the best time for measuring fluorescence should be 2-3 min after adding Fluoro-Development Solution, or based on the color change. When a slight pink color is seen in the wells containing the highest amount of the positive control and the blank well is still clear, the measurement should be carried out.

Thank you for your enquiry and interest regarding ab117129.
I can confirm that the TE buffer specified in the protocol is constituted with 10 mM Tris-HCl pH 7.5-8 and 1 mM EDTA.
If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for contacting Abcam.


Yes, the assay detect 5-mC on both single and double stranded DNA with the nearly same sensitivity. The standard is double stranded DNA.



Please let me know if there is anything else I can help you with.

Thank you for contacting us.

Your credit note ID is ******.

I am sorry that this kit did not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

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I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

Thank you very much for your patience.

We have now received the stimulation graph from the laboratory. Please find it in the attachment. Based on that graph, I am happy to confirm that stimulation at 550nm is absolutely fine, as well as emission reading at 595nm.I hope this information is helpful and does reassure you.

In regards to the inconsistency in your results, I would be pleased to investigate this further. Can you therefore please send me the raw annotated data as well as detailled information on the samples andtheir preparation? Often inconsistency in duplicates are due to technical problems, such as unsufficient washing, bubbles in the well orsmall pipetting difference.I will however be pleasedto look into this problem further with the helpof the data from you.

I look forward to hear back from you. Thank you for your cooperation.

I would like to inform you that we are still investigating the problem you experienced with the kits. The lab has already provided the following information: "The reading can be carried out at 530/590 nm +/- 10 generally. Thus, emission at 595 nm is fine, and excitation at 550 may be Ok but suboptimal". From this Claudiarequested a graph of the excitiation and emission of the fluorochrome used in the kits and we are still waiting for more details. I have sent a reminder to the lab, we are very sorry we cannot still answer this question and I hope we will be able to do so within the next couple of days.

I appreciate the time you have spent on these experiments and would be pleased to arrange a replacement, a refund or credit note in compensation.As a replacement, please note thatthe only lots (for both kits)we currently have in stock are the same as the ones your received, let me know if you would like to receivefree of charge replacements and I will ask the source of the kits ifalternative lotsare available.

I look forward to hearing from you with details of how you would like to proceed.