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

Broad Range dsDNA Qubit

• Followed Qubit protocol

Sample	Reading 1 (ng/ul)	Reading 2(ng/ul)	Average (ng/ul)
S1	184 RFU	-	-
S2	20393 RFU	-	-
18	31	30.8	30.9

D5000 TapeStation

• Followed TapeStation protocol
• Results Link

ColonyID	Collection-Date	Bleach-	Extraction-ID	Index Pair (i5, i7)	i7 bases	i5 bases
M-4 2019-12-04 Non-Bleach 20210225	18	36	GATTCTGC	GACGAGAG