We have planned to sequence a PCR product at MiSeq platform. after 1st step of PCR we perform clean up step. The yield after clean up is < or = to 1ng/ul. Kindly add your advice in respect that should we proceed to index PCR which comprises of only 8 cycles?
Hi,
it is not clear which type of library preparation also indexing you are using. And what you would like to sequence (genome, amlicons..)
Could you tell us it in more detail?
Hi,
I usually performed a nested PCR before indexing in order to increase my PCR amplicon. I used to check only presence/absence of the amplicon on gel agar but never quantified.
Thanks, Dear Marcela Krutova we are following the 16S Metagenomic Sequencing Library Preparation Manual for Illumina MiSeq System. 1st step is Amplicon PCR, 2nd Step is PCR Clean-Up and the 3rd step is Index PCR. Hope you got my question.
Dear Ghulam Mujtaba,
according instruction you have started with DNA concentration 5 ng/ul (2.5ul) =12.5 ng and PCR contained 25 cycles.
Unfortunately, I think that your concentration after first cleanup step is too low.
What instrument did you use for measuring of DNA concentration (input also output - PCR product concentration)?
There is optional checking point, to run PCR product on Bioanalyser chip, but I know it is too expensive.
I agree with you about expensiveness of bioanalyser chip Ms Marcela Krutova, we had use Qubit colorimeter with high sensitivity Qubit kit. we have used 5ng/ul for Amplicon PCR. Afterward the bands are visible and good, we assumed that we didn't need to quantify the amplicon product. The first image(040317) is after Amplicon PCR and the image (07032017) is after cleanup PCR and the quantification revealed that the quantity after clean-up PCR is of all samples is equal to or less 1ng/ul by Qubit.
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