[MonaE]

I have measured the samples after the PCR and they had good measurement especially with gene B. After the beads some of them their concentration even increased. The PCR is a custom panel primers that are ready designed and have been tested before and worked very well.

[addyblanch]

If you can post the concentrations before and after clean up.

[addyblanch]

Also you said your pre/post clean up volumes were the same you're eluting into 25ul. 5ul more than the PCR reaction. This would account for a ng/ul concentration difference but not total. The minimum suggested volume for SPRI elution is also 30ul.

[MonaE]

For example one of the samples was 52 ng/ul after PCR then for gene A after purification it changed to be 18,64 ng/ul. another sample was 22 ng/ul after AMPURe became 33,4 ng/ul however when I repeated the measurement now it is 8,92 after AmPure. The other problem is that the differences between the concentration in different samples is so variable between samples and with different genes. Example sample 9 is 17.38 ng/ul with gene A and 1.542 ng/ul in gene B. Could I still pool them? or run them in different chips?

[addyblanch]

Your ng/ul will be different pre and post clean up because of the volume difference. You have a 20ul PCR reaction and a 25ul clean up elution.

PCR won't amplify the same with different primers!