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Using the Zymo Pico Methyl Seq Library Prep Kit For 1 Re-Do Montipora DNA Samples from KBay Project
Goal Re-do sample 18 library prep, resuspend final libraries in a larger volume Results Prep worked perfectly this time Takeaways I increased all final wash spin times to 3 minutes to make sure no wash buffer carryover occurred
This library prep followed the closely the protocol for the Zymo Pico Methyl Seq Kit from me. See that protocol for detailed descriptions of each steps. Tables and values specific for this prep are included below.
I used the same dilution for sample 18 as made in the previous post
Bisulfite Conversion
Followed exact steps as in protocol
Put samples in fridge overnight
Post-BS Conversion cleanup 20210521
Followed exact steps as in the protocol, except increasing final wash spin to 3 minutes
Amplification with Prep-Amp Primers
Followed exact steps as in the protocol
Priming
2ul PrepAmp Buffer
1ul PrepAmp Primer
PrepAmp
1ul PrepAmp Buffer
3.75ul PrepAmp PreMix
0.3ul PrepAmp Polymerase
PrepAmp Polymerase addition: 0.3ul
DNA Clean and Concentrator
Followed exact steps as in the protocol, except increasing final wash spin to 3 minutes
First Amplification
Followed exact steps as in the protocol
1st Amp
12.5ul Library Amp Mix
1ul Library Amp Primers
Second DNA Clean and Concentrator
Followed exact steps as in the protocol, except increasing final wash spin to 3 minutes
Second Amplification with Index Primers
Followed exact steps as in the protocol, except I used the KAPA HiFi HotStart Ready Mix instead of the Library Amp Mix and I used only 13ul of it, and that the indexes had been combined so I only used 1ul of the combined indexes
Extraction-ID
DNA Volume
KAPA Mix Volume
Primer Pair
Volume Primer
18
12ul
13ul
36
1ul
1X Bead Clean
Followed exact steps as in protocol
Samples were Qubited immediately so they were put on an ice bucket not frozen yet