Using the Zymo Pico Methyl Seq Library Prep Kit For 16 Montipora DNA Samples from KBay Project
Goal Finish library prep for this project, resuspend final libraries in a larger volume
Results One library did not work, all others were fine
Takeaways As usual if there is any extra volume (only like 3ul this time!) after a DCC elution the prep fails. Others look fine
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.
Dilution of samples to 10ng/ul in 10ul 20210520
| Extraction-ID |
DNA reading Avg (ng/uL) |
Dilution for 100ng |
10mM tris HCl to 10ul |
| 43 |
47.4 |
2.11 |
7.89 |
| 54 |
111.5 |
0.90 |
9.10 |
| 39 |
104 |
0.96 |
9.04 |
| 4 |
14.65 |
6.83 |
3.17 |
| 24 |
68.9 |
1.45 |
8.55 |
| 25 |
31.2 |
3.21 |
6.79 |
| 56 |
81.8 |
1.22 |
8.78 |
| 52 |
74.7 |
1.34 |
8.66 |
| 41 |
28.6 |
3.50 |
6.50 |
| 57 |
65.4 |
1.53 |
8.47 |
| 29 |
63.8 |
1.57 |
8.43 |
| 18 |
31.3 |
3.19 |
6.81 |
| 47 |
80 |
1.25 |
8.75 |
| 45 |
73.5 |
1.36 |
8.64 |
| 58 |
17.6 |
5.68 |
4.32 |
| 59 |
21.3 |
4.69 |
5.31 |
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
Amplification with Prep-Amp Primers
- Followed exact steps as in the protocol
- Priming Master Mix calculations (PMM):
- 2ul PrepAmp Buffer * 16.3 = 32.6ul
- 1ul PrepAmp Primer * 16.3 = 16.3ul
- PrepAmp Master Mix calculations (PAMM):
- 1ul PrepAmp Buffer * 16.3 = 16.3ul
- 3.75ul PrepAmp PreMix * 16.3 = 61.125ul
- 0.3ul PrepAmp Polymerase * 16.3 = 4.86ul
- Dilution calculation of PrepAmp Polymerase to add 0.5ul:
- 0.3 PrepAmp Polymerase * 16.3 = 4.86ul
- 0.2ul DNA elution buffer * 16.3 = 3.24ul
DNA Clean and Concentrator
- Followed exact steps as in the protocol
First Amplification
- Followed exact steps as in the protocol
- 1st Amp Master Mix calculation:
- 12.5ul Library Amp Mix * 16.3 = 203.75ul
- 1ul Library Amp Primers * 16.3 = 16.3ul
Second DNA Clean and Concentrator
- Followed exact steps as in the protocol
- Note, after elution sample 18 had ~3ul extra volume from the elution. This usually causes the prep to fail
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
- Table for components in tubes for amplifications:
| Extraction-ID |
DNA Volume |
KAPA Mix Volume |
Primer Pair |
Volume Primer |
| 43 |
12ul |
13ul |
25 |
1ul |
| 54 |
12ul |
13ul |
26 |
1ul |
| 39 |
12ul |
13ul |
27 |
1ul |
| 4 |
12ul |
13ul |
28 |
1ul |
| 24 |
12ul |
13ul |
29 |
1ul |
| 25 |
12ul |
13ul |
30 |
1ul |
| 56 |
12ul |
13ul |
31 |
1ul |
| 52 |
12ul |
13ul |
32 |
1ul |
| 41 |
12ul |
13ul |
33 |
1ul |
| 57 |
12ul |
13ul |
34 |
1ul |
| 29 |
12ul |
13ul |
35 |
1ul |
| 18 |
12ul |
13ul |
36 |
1ul |
| 47 |
12ul |
13ul |
37 |
1ul |
| 45 |
12ul |
13ul |
38 |
1ul |
| 58 |
12ul |
13ul |
39 |
1ul |
| 59 |
12ul |
13ul |
40 |
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
| Sample |
Reading 1 (ng/ul) |
Reading 2(ng/ul) |
Average (ng/ul) |
| S1 |
180 RFU |
- |
- |
| S2 |
19163 RFU |
- |
- |
| 43 |
38.4 |
38.4 |
38.4 |
| 54 |
31.8 |
31.8 |
31.8 |
| 39 |
39 |
38.8 |
38.9 |
| 4 |
36.8 |
36.6 |
36.7 |
| 24 |
16.9 |
16.9 |
16.9 |
| 25 |
38.2 |
38.4 |
28.3 |
| 56 |
19.8 |
20 |
19.9 |
| 52 |
28.2 |
28.2 |
28.2 |
| 41 |
28.2 |
28.4 |
28.3 |
| 57 |
37.8 |
37.6 |
37.7 |
| 29 |
34 |
34.2 |
34.1 |
| 18 |
too low |
- |
- |
| 47 |
34.8 |
34.8 |
34.8 |
| 45 |
33.8 |
33.6 |
33.7 |
| 58 |
15.5 |
15.6 |
15.55 |
| 59 |
32.8 |
33.2 |
33 |
D5000 TapeStation
Samples and Index Sequences
| ColonyID |
Collection-Date |
Bleach- |
Extraction-ID |
Index Pair (i5, i7) |
i7 bases |
i5 bases |
| M-210 |
2019-07-20 |
Non-Bleach |
43 |
25 |
ACTAAGAT |
AACCGCGG |
| M-4 |
2019-07-20 |
Non-Bleach |
54 |
26 |
GTCGGAGC |
GGTTATAA |
| M-212 |
2019-07-20 |
Non-Bleach |
39 |
27 |
CTTGGTAT |
CCAAGTCC |
| M-11 |
2019-12-04 |
Bleach |
4 |
28 |
TCCAACGC |
TTGGACTT |
| M-220 |
2019-12-04 |
Non-Bleach |
24 |
29 |
CCGTGAAG |
CAGTGGAT |
| M-12 |
2019-12-04 |
Non-Bleach |
25 |
30 |
TTACAGGA |
TGACAAGC |
| M-218 |
2019-07-20 |
Non-Bleach |
56 |
31 |
GGCATTCT |
CTAGCTTG |
| M-211 |
2019-07-20 |
Bleach |
52 |
32 |
AATGCCTC |
TCGATCCA |
| M-3 |
2019-07-20 |
Bleach |
41 |
33 |
TACCGAGG |
CCTGAACT |
| M-19 |
2019-07-20 |
Bleach |
57 |
34 |
CGTTAGAA |
TTCAGGTC |
| M-19 |
2019-12-04 |
Bleach |
29 |
35 |
AGCCTCAT |
AGTAGAGA |
| M-217 |
2019-07-20 |
Bleach |
47 |
37 |
TCGTAGTG |
AGACTTGG |
| M-11 |
2019-07-20 |
Bleach |
45 |
38 |
CTACGACA |
GAGTCCAA |
| M-20 |
2019-07-20 |
Non-Bleach |
58 |
39 |
TAAGTGGT |
CTTAAGCC |
| M-220 |
2019-07-20 |
Non-Bleach |
59 |
40 |
CGGACAAC |
TCCGGATT |
