Project: Thermal Transplant Molecular

Goal

Re-amplify PicoMethyl-seq libraries that had low output (<5ng/ul) from initial prep using this protocol.

Protocol

Repeating the last two steps of this protocol:

1) Second amplification with 4 cycles instead of 10 cycles

2) 1X bead clean up

For all samples, I used the following inputs for the second amplification:

• 12 uL of the PicoMethyl-seq library
• 13 uL of Library Amp Master Mix (2x)
• 1 uL of the combined i5 and i7 index primers (I used the same indices as the original library prep)

Samples, indices, original Qubit quantification, post re-amplification Qubit quantification

Sample #	i5/i7 Index #	Original Library (ng/uL)	Re-amp DNA #1 (ng/uL)	Re-amp DNA #2 (ng/uL)
15	15	LOW	9.56	9.32
17	17	5.81	14.2	13.9
19	19	LOW	LOW	LOW
20	20	3.24	7.12	7.00
21	21	4.69	16.5	16.1
26	26	3.54	19.1	18.6
27	27	5.44	20.6	19.8
28	28	2.86	17.5	17.2
32	32	5.57	LOW	LOW
45	45	5.4	21.2	20.8
Qubit std.1			178.09
Qubit std.2			19930.40

Tapestation Results

D5000 Tapestation protocol

Full Tapestation Report 1

Full Tapestation Report 2