Noi/NOTES/2013-7-19: Difference between revisions
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* From the result, it seem like smaller volume of template show less background and stronger expected products. However, it didn't look as clean as I got from the first test experiment. I suspect that Exo I/III may not completely digest ds/ssDNA. I then tested enzyme activity by incubating equal amount of probes and bis-cvt gDNA I used in capture and I saw very clean gel. This suggested that enzymatic activity was good. I also run check circularized template in TBU gel (loaded 3ul) to see if there is the probe or gDNA left. I did not see either. However by PAGE verification, I may not be able to see the left over of probes and gDNA if the level was very low. | * From the result, it seem like smaller volume of template show less background and stronger expected products. However, it didn't look as clean as I got from the first test experiment. I suspect that Exo I/III may not completely digest ds/ssDNA. I then tested enzyme activity by incubating equal amount of probes and bis-cvt gDNA I used in capture and I saw very clean gel. This suggested that enzymatic activity was good. I also run check circularized template in TBU gel (loaded 3ul) to see if there is the probe or gDNA left. I did not see either. However by PAGE verification, I may not be able to see the left over of probes and gDNA if the level was very low. | ||
[[File:ZhangLab_2 2013-07-22 15hr 11min_confirmExoI-III-digestedprobes.jpg| | [[File:ZhangLab_2 2013-07-22 15hr 11min_confirmExoI-III-digestedprobes.jpg| 350px]] [[File:ZhangLab_2 2013-07-23 12hr 59min_TBU_circularized DNA.jpg| 110px]] | ||
Gel1 = ExoI/III with equal amount of probe and bis-cvt gDNA (loaded difeerent amount) | |||
Gel2 = 3ul of NTC and well B5 circularized DNA | |||
* I decided to used 4% of template and perform PCR amplification in total 100ul reaction to make sequencing libraries. | * I decided to used 4% of template and perform PCR amplification in total 100ul reaction to make sequencing libraries. | ||
{| {{table}} | {| {{table}} | ||
Line 441: | Line 444: | ||
* Performed PAGE quantification by loading sample 2ul | * Performed PAGE quantification by loading sample 2ul | ||
[[File:ZhangLab_2 2013-07-23 17hr 11min_qPCR_BP-REAL_A1-H1.jpg| 400px]] [[File:ZhangLab_2 2013-07-23 17hr 11min_qPCR_BP-REAL_A2-H2.jpg| 400px]] | [[File:ZhangLab_2 2013-07-23 17hr 11min_qPCR_BP-REAL_A1-H1.jpg| 400px]] [[File:ZhangLab_2 2013-07-23 17hr 11min_qPCR_BP-REAL_A2-H2.jpg| 400px]] | ||
[[File:ZhangLab_2 2013-07-23 17hr 59min_qPCR_BP-REAL_A3-H3.jpg| 400px]] [[File:ZhangLab_2 2013-07-23 17hr 59min_qPCR_BP-REAL_A4-B5.jpg| | |||
[[File:ZhangLab_2 2013-07-23 17hr 59min_qPCR_BP-REAL_A3-H3.jpg| 400px]] [[File:ZhangLab_2 2013-07-23 17hr 59min_qPCR_BP-REAL_A4-B5.jpg| 480px]] | |||
==== PAGE quantification ==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Well IDs''' | |||
| align="center" style="background:#f0f0f0;"|'''Index''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Yields in 30ul (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Well IDs''' | |||
| align="center" style="background:#f0f0f0;"|'''Pool1 volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount''' | |||
| align="center" style="background:#f0f0f0;"|'''Pool2 volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount (ng)''' | |||
|- | |||
| A1||Index 57||6.17||185.16||A1||8.10||50.00||NA||NA | |||
|- | |||
| B1||Index 58||6.21||186.45||B1||8.05||50.00||NA||NA | |||
|- | |||
| C1||Index 59||6.42||192.60||C1||7.79||50.00||NA||NA | |||
|- | |||
| D1||Index 60||6.20||186.10||D1||8.06||50.00||NA||NA | |||
|- | |||
| E1||Index 61||6.32||189.72||E1||7.91||50.00||NA||NA | |||
|- | |||
| F1||Index 62||6.61||198.32||F1||7.56||50.00||NA||NA | |||
|- | |||
| G1||Index 63||6.65||199.44||G1||7.52||50.00||NA||NA | |||
|- | |||
| H1||Index 64||5.93||177.79||H1||8.44||50.00||NA||NA | |||
|- | |||
| A2||Index 65||4.94||148.24||A2||10.12||50.00||NA||NA | |||
|- | |||
| B2||Index 66||5.54||166.12||B2||9.03||50.00||NA||NA | |||
|- | |||
| C2||Index 67||4.93||147.94||C2||10.14||50.00||NA||NA | |||
|- | |||
| D2||Index 68||5.56||166.68||D2||9.00||50.00||NA||NA | |||
|- | |||
| E2||Index 69||4.53||135.81||E2||11.04||50.00||NA||NA | |||
|- | |||
| F2||Index 70||5.39||161.60||F2||9.28||50.00||NA||NA | |||
|- | |||
| G2||Index 71||4.99||149.63||G2||10.02||50.00||NA||NA | |||
|- | |||
| H2||Index 72||5.61||168.28||H2||8.91||50.00||NA||NA | |||
|- | |||
| A3||Index 73||1.47||44.22||A3||NA||NA||12.21||18.00 | |||
|- | |||
| B3||Index 74||2.11||63.34||B3||23.68||50.00||NA||NA | |||
|- | |||
| C3||Index 75||2.81||84.45||C3||17.76||50.00||NA||NA | |||
|- | |||
| D3||Index 76||3.68||110.48||D3||13.58||50.00||NA||NA | |||
|- | |||
| E3||Index 77||3.92||117.57||E3||12.76||50.00||NA||NA | |||
|- | |||
| F3||Index 78||3.61||108.40||F3||13.84||50.00||NA||NA | |||
|- | |||
| G3||Index 79||3.68||110.29||G3||13.60||50.00||NA||NA | |||
|- | |||
| H3||Index 80||3.67||110.14||H3||13.62||50.00||NA||NA | |||
|- | |||
| A4||Index 81||0.62||18.67||A4||NA||NA||28.93||18.00 | |||
|- | |||
| B4||Index 82||5.31||159.39||B4||9.41||50.00||NA||NA | |||
|- | |||
| C4||Index 83||5.10||153.05||C4||9.80||50.00||NA||NA | |||
|- | |||
| D4||Index 84||5.88||176.34||D4||8.51||50.00||NA||NA | |||
|- | |||
| E4||Index 85||5.85||175.36||E4||8.55||50.00||NA||NA | |||
|- | |||
| F4||Index 86||5.89||176.80||F4||8.48||50.00||NA||NA | |||
|- | |||
| G4||Index 87||5.89||176.78||G4||8.49||50.00||NA||NA | |||
|- | |||
| H4||Index 88||5.46||163.76||H4||9.16||50.00||NA||NA | |||
|- | |||
| A5||Index 89||5.24||157.26||A5||9.54||50.00||NA||NA | |||
|- | |||
| B5||Index 90||4.52||135.63||B5||11.06||50.00||NA||NA | |||
|} | |||
* There were two samples have very low concentration, so I pool these two libraries together and purified in on lane of 5-well TBE gel | |||
* The rest of libraries were pooled together and purified in 2 of 5-well TBE gels. | |||
==== PAGE size-selection ==== | |||
[[File:ZhangLab_2 2013-07-23 21hr 32min_BP-SeqLib-PAGE-SS.jpg| 400px]] | [[File:ZhangLab_2 2013-07-23 21hr 32min_BP-SeqLib-PAGE-SS.jpg| 400px]] |
Revision as of 20:54, 29 July 2013
DNA methylation assay for Blueprint project
Bisulfite conversion
I quantified gDNA concentration by Qubit dsDNA assay. Note that the volume of each sample they sent us was variied so I can not summarize total amount of each sample. However, based on concentration by Qubit the total amount we got of each sample less than 1ug as they claimed. Therefore I may not have enough gDNA for the second trial even I used only 500ng for bisulfite conversion. Also, there were few samples which total amount is very low so total amount for bis-cvt less than 300ng.
- I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
Sample IDs | WellIDs | Conc. (ng/ul) | Volume (ul) | H2O (ul) | CT conversion reagent (ul) | Total volume (ul) | Total amount (ng) |
1 | A1 | 37.71 | 13.26 | 6.74 | 130.00 | 150.00 | 500.00 |
2 | B1 | 39.43 | 12.68 | 7.32 | 130.00 | 150.00 | 500.00 |
3 | C1 | 50.57 | 9.89 | 10.11 | 130.00 | 150.00 | 500.00 |
4 | D1 | 34.86 | 14.34 | 5.66 | 130.00 | 150.00 | 500.00 |
5 | E1 | 56.00 | 8.93 | 11.07 | 130.00 | 150.00 | 500.00 |
6 | F1 | 44.29 | 11.29 | 8.71 | 130.00 | 150.00 | 500.00 |
7 | G1 | 73.71 | 6.78 | 13.22 | 130.00 | 150.00 | 500.00 |
8 | H1 | 29.71 | 16.83 | 3.17 | 130.00 | 150.00 | 500.00 |
9 | A2 | 40.29 | 12.41 | 7.59 | 130.00 | 150.00 | 500.00 |
10 | B2 | 33.43 | 14.96 | 5.04 | 130.00 | 150.00 | 500.00 |
11 | C2 | 44.86 | 11.15 | 8.85 | 130.00 | 150.00 | 500.00 |
12 | D2 | 52.86 | 9.46 | 10.54 | 130.00 | 150.00 | 500.00 |
**13 | E2 | 11.34 | 9.00 | 11.00 | 130.00 | 150.00 | 102.09 |
*14 | F2 | 36.86 | 9.00 | 11.00 | 130.00 | 150.00 | 331.71 |
**15 | G2 | 26.74 | 9.00 | 11.00 | 130.00 | 150.00 | 240.69 |
*16 | H2 | 50.00 | 9.00 | 11.00 | 130.00 | 150.00 | 450.00 |
17 | A3 | 34.00 | 14.71 | 5.29 | 130.00 | 150.00 | 500.00 |
18 | B3 | 31.14 | 16.06 | 3.94 | 130.00 | 150.00 | 500.00 |
19 | C3 | 26.46 | 18.90 | 1.10 | 130.00 | 150.00 | 500.00 |
*20 | D3 | 20.29 | 20.00 | 0.00 | 130.00 | 150.00 | 405.71 |
*21 | E3 | 18.23 | 20.00 | 0.00 | 130.00 | 150.00 | 364.57 |
22 | F3 | 29.43 | 16.99 | 3.01 | 130.00 | 150.00 | 500.00 |
23 | G3 | 47.71 | 10.48 | 9.52 | 130.00 | 150.00 | 500.00 |
24 | H3 | 32.57 | 15.35 | 4.65 | 130.00 | 150.00 | 500.00 |
25 | A4 | 29.14 | 17.16 | 2.84 | 130.00 | 150.00 | 500.00 |
*26 | B4 | 19.60 | 20.00 | 0.00 | 130.00 | 150.00 | 392.00 |
27 | C4 | 24.31 | 20.00 | 0.00 | 130.00 | 150.00 | 486.29 |
28 | D4 | 23.66 | 20.00 | 0.00 | 130.00 | 150.00 | 473.14 |
29 | E4 | 47.43 | 10.54 | 9.46 | 130.00 | 150.00 | 500.00 |
30 | F4 | 76.86 | 6.51 | 13.49 | 130.00 | 150.00 | 500.00 |
31 | G4 | 40.00 | 12.50 | 7.50 | 130.00 | 150.00 | 500.00 |
32 | H4 | 32.29 | 15.49 | 4.51 | 130.00 | 150.00 | 500.00 |
33 | A5 (12878) | 58.00 | 8.62 | 11.38 | 130.00 | 150.00 | 500.00 |
34 | B5 (12878) | 59.00 | 8.47 | 11.53 | 131.00 | 151.00 | 500.00 |
Program
1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold
- Eluted bisulfite converted DNA form the bead with 20ul H2O
- I used 1ul for ssDNA Qubit assay (200x dilution). The volume left after assay ~15ul.
ssDNA Qubit assay result
Sample IDs | WellIDs | Start amount (ng) | Conc. (ng/ul) | Yield in 17ul (ng) | Yield left after Qubit (15ul) |
% Recovery in 17ul |
1 | A1 | 500.00 | 21.00 | 357.00 | 315.00 | 71.40 |
2 | B1 | 500.00 | 24.40 | 414.80 | 366.00 | 82.96 |
3 | C1 | 500.00 | 19.10 | 324.70 | 286.50 | 64.94 |
4 | D1 | 500.00 | 22.60 | 384.20 | 339.00 | 76.84 |
5 | E1 | 500.00 | 20.60 | 350.20 | 309.00 | 70.04 |
6 | F1 | 500.00 | 21.20 | 360.40 | 318.00 | 72.08 |
7 | G1 | 500.00 | 22.00 | 374.00 | 330.00 | 74.80 |
8 | H1 | 500.00 | 22.20 | 377.40 | 333.00 | 75.48 |
9 | A2 | 500.00 | 20.60 | 350.20 | 309.00 | 70.04 |
10 | B2 | 500.00 | 24.00 | 408.00 | 360.00 | 81.60 |
11 | C2 | 500.00 | 19.06 | 324.02 | 285.90 | 64.80 |
12 | D2 | 500.00 | 18.78 | 319.26 | 281.70 | 63.85 |
13 | E2 | 102.09 | 2.06 | 35.02 | 30.90 | 34.30 |
14 | F2 | 331.71 | 8.08 | 137.36 | 121.20 | 41.41 |
15 | G2 | 240.69 | 6.50 | 110.50 | 97.50 | 45.91 |
16 | H2 | 450.00 | 12.32 | 209.44 | 184.80 | 46.54 |
17 | A3 | 500.00 | 11.56 | 196.52 | 173.40 | 39.30 |
18 | B3 | 500.00 | 12.24 | 208.08 | 183.60 | 41.62 |
19 | C3 | 500.00 | 11.44 | 194.48 | 171.60 | 38.90 |
20 | D3 | 405.71 | 7.92 | 134.64 | 118.80 | 33.19 |
21 | E3 | 364.57 | 8.10 | 137.70 | 121.50 | 37.77 |
22 | F3 | 500.00 | 11.38 | 193.46 | 170.70 | 38.69 |
23 | G3 | 500.00 | 13.96 | 237.32 | 209.40 | 47.46 |
24 | H3 | 500.00 | 19.72 | 335.24 | 295.80 | 67.05 |
25 | A4 | 500.00 | 21.20 | 360.40 | 318.00 | 72.08 |
26 | B4 | 392.00 | 23.00 | 391.00 | 345.00 | 99.74 |
27 | C4 | 486.29 | 23.80 | 404.60 | 357.00 | 83.20 |
28 | D4 | 473.14 | 31.40 | 533.80 | 471.00 | 112.82 |
29 | E4 | 500.00 | 20.80 | 353.60 | 312.00 | 70.72 |
30 | F4 | 500.00 | 10.46 | 177.82 | 156.90 | 35.56 |
31 | G4 | 500.00 | 47.00 | 799.00 | 705.00 | 159.80 |
32 | H4 | 500.00 | 56.00 | 952.00 | 840.00 | 190.40 |
33 | A5 (12878) | 500.00 | 24.00 | 408.00 | 360.00 | 81.60 |
34 | B5 (12878) | 500.00 | 24.60 | 418.20 | 369.00 | 83.64 |
- There are few sample have %recovery >100%. I repeat measurement again and got the same concentration. This is possible that I used too small volume when assay gDNA concentration, and there is some error of pipetting.
- Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion.
BSPP capture set up
Original set
- Conc.: 25.4 ng/ul (by Qubit ssDNA assay)
Probe:target | 500:1 | ' |
Probe size | 12000 | |
Template | 150 | ng |
Human gDNA MW | 1.9500E+12 | g/mol |
Human gDNA (150ng) | 7.6923E-20 | mol (150ng/1.9500E+12 g/mol) |
Probe required (500:1) | 3.8462E-17 | mol (7.6923E-20 mol * 500) |
Probe MW (12,000 probes, 110nt) | 4.2900E+08 | g/mol (12000 probes * 110nt * 325Da/nt) |
Amount probe required | 1.6500E-08 | g (4.2900E+08 g/mol * 3.8462E-17 mol) |
Amount probe required | 16.50 | ng |
Subset A
- Conc.: 17.6ng/ul (by Qubit ssDNA assay)
Probe:target | 500:1 | ' |
Probe size | 566 | probes |
Template | 150 | ng |
Human gDNA MW | 1.9500E+12 | g/mol (3E+09 * 650Da/bp+157.9Da) |
Human gDNA (200ng) | 7.6923E-20 | mol (200ng/1.9500E+12 g/mol) |
Probe required (500:1) | 3.8462E-17 | mol (1.5385E-19mol * 500) |
Probe MW (566 probes, 1100nt) | 2.0235E+07 | g/mol (566 probes * 110nt * 325Da/nt) |
Amount probe required | 7.7825E-10 | g 2.0235E+07 g/mol * 3.8462E-17mol) |
Amount probe required | 0.78 | ng |
- So for 1:5:5 (Original:A:B), amount probe required = 0.78ng * 5 = 8.89ng
Subset B
- Conc.: 57.9ng/ul (by Qubit ssDNA assay)
Probe:target | 500:1 | ' |
Probe size | 11397 | probes |
Template | 150 | ng |
Human gDNA MW | 1.9500E+12 | g/mol (3E+09 * 650Da/bp+157.9Da) |
Human gDNA (200ng) | 7.6923E-20 | mol (150ng/1.9500E+12 g/mol) |
Probe required (500:1) | 3.8462E-17 | mol (1.5385E-19mol * 500) |
Probe MW (11,397 probes, 1100nt) | 4.0744E+08 | g/mol (11397 probes * 110nt * 325Da/nt) |
Amount probe required | 1.5671E-08 | g (4.0744E+08 g/mol * 3.8462E-17 mol) |
Amount probe required | 15.67 | ng |
- So for 1:5:5 (Original:A:B), amount probe required = 15.67ng * 5 = 78.35ng
Summary of probes required for cpature
Probe set | Conc. (ng/ul) | Vmount required | Volume (ul)/samples | Volume for ~38x rxn |
Original | 25.40 | 16.50 | 0.65 | 24.69 |
Subset A | 17.60 | 3.89 | 0.22 | 8.40 |
Subset B | 57.90 | 78.35 | 1.35 | 51.42 |
- I will do two captures for positive control (12878) to see the consistency between the two technical replicates and one NTC
- I will save at least 2ul of bis-cvt DNA for bisulfite PCR if we need to do that. However, Dr. Zhang suggested us to ignore the recommended_4 site.
Sample IDs | WellIDs | Conc. (ng/ul) | Volume required (ul) | BP-O (ul) | BP.norm-A (ul) | BP.norm-B (ul) | 10X Ampligase Buffer (ul) |
H2O (ul) | Total vulome (ul) | Total biscvt DNA (ng) |
1 | A1 | 21.00 | 7.14 | 0.65 | 0.22 | 1.35 | 2.00 | 8.64 | 20.00 | 150.00 |
2 | B1 | 24.40 | 6.15 | 0.65 | 0.22 | 1.35 | 2.00 | 9.63 | 20.00 | 150.00 |
3 | C1 | 19.10 | 7.85 | 0.65 | 0.22 | 1.35 | 2.00 | 7.93 | 20.00 | 150.00 |
4 | D1 | 22.60 | 6.64 | 0.65 | 0.22 | 1.35 | 2.00 | 9.14 | 20.00 | 150.00 |
5 | E1 | 20.60 | 7.28 | 0.65 | 0.22 | 1.35 | 2.00 | 8.50 | 20.00 | 150.00 |
6 | F1 | 21.20 | 7.08 | 0.65 | 0.22 | 1.35 | 2.00 | 8.70 | 20.00 | 150.00 |
7 | G1 | 22.00 | 6.82 | 0.65 | 0.22 | 1.35 | 2.00 | 8.96 | 20.00 | 150.00 |
8 | H1 | 22.20 | 6.76 | 0.65 | 0.22 | 1.35 | 2.00 | 9.02 | 20.00 | 150.00 |
9 | A2 | 20.60 | 7.28 | 0.65 | 0.22 | 1.35 | 2.00 | 8.50 | 20.00 | 150.00 |
10 | B2 | 24.00 | 6.25 | 0.65 | 0.22 | 1.35 | 2.00 | 9.53 | 20.00 | 150.00 |
11 | C2 | 19.06 | 7.87 | 0.65 | 0.22 | 1.35 | 2.00 | 7.91 | 20.00 | 150.00 |
12 | D2 | 18.78 | 7.99 | 0.65 | 0.22 | 1.35 | 2.00 | 7.79 | 20.00 | 150.00 |
13 | E2 | 2.06 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 26.78 |
14 | F2 | 8.08 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 105.04 |
15 | G2 | 6.50 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 84.50 |
16 | H2 | 12.32 | 12.18 | 0.65 | 0.22 | 1.35 | 2.00 | 3.60 | 20.00 | 150.00 |
17 | A3 | 11.56 | 12.98 | 0.65 | 0.22 | 1.35 | 2.00 | 2.80 | 20.00 | 150.00 |
18 | B3 | 12.24 | 12.25 | 0.65 | 0.22 | 1.35 | 2.00 | 3.53 | 20.00 | 150.00 |
19 | C3 | 11.44 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 148.72 |
20 | D3 | 7.92 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 102.96 |
21 | E3 | 8.10 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 105.30 |
22 | F3 | 11.38 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 147.94 |
23 | G3 | 13.96 | 10.74 | 0.65 | 0.22 | 1.35 | 2.00 | 5.04 | 20.00 | 150.00 |
24 | H3 | 19.72 | 7.61 | 0.65 | 0.22 | 1.35 | 2.00 | 8.17 | 20.00 | 150.00 |
25 | A4 | 21.20 | 7.08 | 0.65 | 0.22 | 1.35 | 2.00 | 8.70 | 20.00 | 150.00 |
26 | B4 | 23.00 | 6.52 | 0.65 | 0.22 | 1.35 | 2.00 | 9.26 | 20.00 | 150.00 |
27 | C4 | 23.80 | 6.30 | 0.65 | 0.22 | 1.35 | 2.00 | 9.48 | 20.00 | 150.00 |
28 | D4 | 31.40 | 4.78 | 0.65 | 0.22 | 1.35 | 2.00 | 11.00 | 20.00 | 150.00 |
29 | E4 | 20.80 | 7.21 | 0.65 | 0.22 | 1.35 | 2.00 | 8.57 | 20.00 | 150.00 |
30 | F4 | 10.46 | 13.00 | 0.65 | 0.22 | 1.35 | 2.00 | 2.78 | 20.00 | 135.98 |
31 | G4 | 47.00 | 3.19 | 0.65 | 0.22 | 1.35 | 2.00 | 12.59 | 20.00 | 150.00 |
32 | H4 | 56.00 | 2.68 | 0.65 | 0.22 | 1.35 | 2.00 | 13.10 | 20.00 | 150.00 |
33 | A5 (12878) | 24.00 | 6.25 | 0.65 | 0.22 | 1.35 | 2.00 | 9.53 | 20.00 | 150.00 |
34 | B5 (12878) | 24.60 | 6.10 | 0.65 | 0.22 | 1.35 | 2.00 | 9.68 | 20.00 | 150.00 |
35 | C5 (NTC) | 0.00 | 0.00 | 0.65 | 0.22 | 1.35 | 2.00 | 15.78 | 20.00 | 0.00 |
Reaction mix
Components | 1x rxn | 38 rxn mix |
10X Ampligase buffer | 2.00 | 76.00 |
BP-O | 0.65 | 24.70 |
BP.norm-A | 0.22 | 8.36 |
BP.norm-B | 1.35 | 51.30 |
Total | 4.22 | 160.36 |
- Aliquot ~18ul to 8-tube strip and add 4.22ul of reaction mix to the 96-well plate with bis-cvt DNA and H2O with multichannel pipette
- Mix 15 time by pipetting
- Add 40ul of mineral oil & seal with silicone sealing mat
- Spin the plate and place on thermocycler
Program -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2ul SLN mix (10x Titanium Taq; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN mix solution
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume (ul), total 80ul |
Titanium Taq (Klentaq fragment) | 10 | U/ul | 2 | U/ul | 16.00 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 8.00 |
dNTP | 1 | mM | 100 | uM | 8.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 8.00 |
H2O | 40.00 |
- Aliquot 9ul to 8-tube strip and add 2ul to each well with multichannel pipette.
- Note: Actually, Clontech doesn't provide the concentration of Titanium Taq. They only said 50X concentration. I assume it has the same concentration as Stoffel fragment.
PCR Amplification (TEST)
- Before amplify with barcode primer I test if the capture work by doing PCR in small volume (25ul) with 2ul of circularized DNA
Components | 1x rxn | 30 x rxn mix |
Captured template | 2.00 | 0.00 |
10uM AmpF6.4Sol | 0.50 | 15.00 |
10uM AmpR6.3.Ind1 | 0.50 | 15.00 |
2X KAPA SYBR MM | 12.50 | 375.00 |
H2O | 9.50 | 285.00 |
Total | 25.00 |
- Aliquot 23ul, add 2ul of circularized DNA template
Program (Eppendorf Realplex 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
- Loaded 3ul of PCR product in TBE gel
File:ZhangLab 2 2013-07-22 19hr 23min TEST-BP-REAL.jpg
- It seem like I din't see very clear expected bands between 400-480bp, and the background is very high. I wonder if if added too much template, but for previous experiment I add 10ul of template in total 50ul, and got very clear bands.
- I TEST PCR again by varying template volume (0.5, 1, 2, and 4ul in total 25ul) to test if the volume of template interfere PCR.
- From the result, it seem like smaller volume of template show less background and stronger expected products. However, it didn't look as clean as I got from the first test experiment. I suspect that Exo I/III may not completely digest ds/ssDNA. I then tested enzyme activity by incubating equal amount of probes and bis-cvt gDNA I used in capture and I saw very clean gel. This suggested that enzymatic activity was good. I also run check circularized template in TBU gel (loaded 3ul) to see if there is the probe or gDNA left. I did not see either. However by PAGE verification, I may not be able to see the left over of probes and gDNA if the level was very low.
File:ZhangLab 2 2013-07-22 15hr 11min confirmExoI-III-digestedprobes.jpg File:ZhangLab 2 2013-07-23 12hr 59min TBU circularized DNA.jpg Gel1 = ExoI/III with equal amount of probe and bis-cvt gDNA (loaded difeerent amount) Gel2 = 3ul of NTC and well B5 circularized DNA
- I decided to used 4% of template and perform PCR amplification in total 100ul reaction to make sequencing libraries.
Components | 1x rxn | 38 rxn mix |
Captured template | 4.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 76.00 |
10uM AmpR6.3Sol | 2.00 | 0.00 |
2X KAPA SYBR MM | 50.00 | 1900.00 |
H2O | 42.00 | 1596.00 |
Total | 100.00 |
Program (Eppendorf Realplex 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
- Purified PCR product with 0.7 volume Ampure beads (I dilute bead 1:1 with 20% PEG/5M NaCl before adding 0.7 volume for purification).
- Eluted with 30ul H2O
- Performed PAGE quantification by loading sample 2ul
File:ZhangLab 2 2013-07-23 17hr 11min qPCR BP-REAL A1-H1.jpg File:ZhangLab 2 2013-07-23 17hr 11min qPCR BP-REAL A2-H2.jpg File:ZhangLab 2 2013-07-23 17hr 59min qPCR BP-REAL A3-H3.jpg File:ZhangLab 2 2013-07-23 17hr 59min qPCR BP-REAL A4-B5.jpg
PAGE quantification
Well IDs | Index | Conc. (ng/ul) | Yields in 30ul (ng) | Well IDs | Pool1 volume (ul) | Amount | Pool2 volume (ul) | Amount (ng) |
A1 | Index 57 | 6.17 | 185.16 | A1 | 8.10 | 50.00 | NA | NA |
B1 | Index 58 | 6.21 | 186.45 | B1 | 8.05 | 50.00 | NA | NA |
C1 | Index 59 | 6.42 | 192.60 | C1 | 7.79 | 50.00 | NA | NA |
D1 | Index 60 | 6.20 | 186.10 | D1 | 8.06 | 50.00 | NA | NA |
E1 | Index 61 | 6.32 | 189.72 | E1 | 7.91 | 50.00 | NA | NA |
F1 | Index 62 | 6.61 | 198.32 | F1 | 7.56 | 50.00 | NA | NA |
G1 | Index 63 | 6.65 | 199.44 | G1 | 7.52 | 50.00 | NA | NA |
H1 | Index 64 | 5.93 | 177.79 | H1 | 8.44 | 50.00 | NA | NA |
A2 | Index 65 | 4.94 | 148.24 | A2 | 10.12 | 50.00 | NA | NA |
B2 | Index 66 | 5.54 | 166.12 | B2 | 9.03 | 50.00 | NA | NA |
C2 | Index 67 | 4.93 | 147.94 | C2 | 10.14 | 50.00 | NA | NA |
D2 | Index 68 | 5.56 | 166.68 | D2 | 9.00 | 50.00 | NA | NA |
E2 | Index 69 | 4.53 | 135.81 | E2 | 11.04 | 50.00 | NA | NA |
F2 | Index 70 | 5.39 | 161.60 | F2 | 9.28 | 50.00 | NA | NA |
G2 | Index 71 | 4.99 | 149.63 | G2 | 10.02 | 50.00 | NA | NA |
H2 | Index 72 | 5.61 | 168.28 | H2 | 8.91 | 50.00 | NA | NA |
A3 | Index 73 | 1.47 | 44.22 | A3 | NA | NA | 12.21 | 18.00 |
B3 | Index 74 | 2.11 | 63.34 | B3 | 23.68 | 50.00 | NA | NA |
C3 | Index 75 | 2.81 | 84.45 | C3 | 17.76 | 50.00 | NA | NA |
D3 | Index 76 | 3.68 | 110.48 | D3 | 13.58 | 50.00 | NA | NA |
E3 | Index 77 | 3.92 | 117.57 | E3 | 12.76 | 50.00 | NA | NA |
F3 | Index 78 | 3.61 | 108.40 | F3 | 13.84 | 50.00 | NA | NA |
G3 | Index 79 | 3.68 | 110.29 | G3 | 13.60 | 50.00 | NA | NA |
H3 | Index 80 | 3.67 | 110.14 | H3 | 13.62 | 50.00 | NA | NA |
A4 | Index 81 | 0.62 | 18.67 | A4 | NA | NA | 28.93 | 18.00 |
B4 | Index 82 | 5.31 | 159.39 | B4 | 9.41 | 50.00 | NA | NA |
C4 | Index 83 | 5.10 | 153.05 | C4 | 9.80 | 50.00 | NA | NA |
D4 | Index 84 | 5.88 | 176.34 | D4 | 8.51 | 50.00 | NA | NA |
E4 | Index 85 | 5.85 | 175.36 | E4 | 8.55 | 50.00 | NA | NA |
F4 | Index 86 | 5.89 | 176.80 | F4 | 8.48 | 50.00 | NA | NA |
G4 | Index 87 | 5.89 | 176.78 | G4 | 8.49 | 50.00 | NA | NA |
H4 | Index 88 | 5.46 | 163.76 | H4 | 9.16 | 50.00 | NA | NA |
A5 | Index 89 | 5.24 | 157.26 | A5 | 9.54 | 50.00 | NA | NA |
B5 | Index 90 | 4.52 | 135.63 | B5 | 11.06 | 50.00 | NA | NA |
- There were two samples have very low concentration, so I pool these two libraries together and purified in on lane of 5-well TBE gel
- The rest of libraries were pooled together and purified in 2 of 5-well TBE gels.
PAGE size-selection
File:ZhangLab 2 2013-07-23 21hr 32min BP-SeqLib-PAGE-SS.jpg