New member and new to running my own MiSeq Sequencer!
We are trying to figure out how to modify TruSeq PCR free DNA library prep kit protocol to make a library with our pool of barcoded amplicons (16s and ITS) for sequencing on the MiSeq. Has anyone else done this or seen any published protocols combining these methods?
The problem I am running into is that we use SequalPrep Plates to normalized our PCR amplicons (~350bp) and then pool. This theoretically provides us with 1.9ml of pooled PCR product at ~1ng/ul (we ended up with about 0.7 bc of negtive controls and failed PCR reactions).
We would start the TruSeq protocol with the dAtailing step, which calls for 15ul of sample that I guess is around 800ng since you start the full protocol with 1100 (for a 350bp insert).
Speed vac to concentrate would be problematic due to the elution buffer used in SequalPrep. I tried a Zymo clean and concentrator column, but lost 60% of the yield. SequalPrep does have a sequential elution recommendation, which I have not tried yet, but at 5 min per well it will be time limiting. Any suggestions?
We are trying to figure out how to modify TruSeq PCR free DNA library prep kit protocol to make a library with our pool of barcoded amplicons (16s and ITS) for sequencing on the MiSeq. Has anyone else done this or seen any published protocols combining these methods?
The problem I am running into is that we use SequalPrep Plates to normalized our PCR amplicons (~350bp) and then pool. This theoretically provides us with 1.9ml of pooled PCR product at ~1ng/ul (we ended up with about 0.7 bc of negtive controls and failed PCR reactions).
We would start the TruSeq protocol with the dAtailing step, which calls for 15ul of sample that I guess is around 800ng since you start the full protocol with 1100 (for a 350bp insert).
Speed vac to concentrate would be problematic due to the elution buffer used in SequalPrep. I tried a Zymo clean and concentrator column, but lost 60% of the yield. SequalPrep does have a sequential elution recommendation, which I have not tried yet, but at 5 min per well it will be time limiting. Any suggestions?
Comment