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Old 06-05-2020, 01:19 AM   #3
Saurabh_Raj
Junior Member
 
Location: Leipzig

Join Date: May 2020
Posts: 2
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Hi,
Thank you for your message. We have access to discarded flowcells to test our protocol. Let me explain a bit further. The discarded PE sequence that we obtain from Illumina have sequence in the order:
3'-P7-Index-Read2-sequenced DNA-Read1-P5-5'
Once the flow cell is denatured to remove any residual fluorophore from sequencing, we should get the above mentioned ssDNA. I use Read2 complimentary as the regeneration primer. When the regeneration is done, complimentary of Read1 - Read1*Cy3 is injected to see whether regeneration was successful or not. When regeneration happens, Read1 region will be annealed with regenerated strand, as you mentioned. In that case, Read1*Cy3 should not bind to the regenerated strands, isn't it? But I still see binding as strong as that for ssDNA.

I have used the labeled Read2 extension primer. But it is hard to say whether extension happened or not as the primer will remain hybridized to the ssDNA even when extension fails. I am thinking to implement the usage of labeled dNTPs next.

Best regards,
Saurabh
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