Noi/NOTES/2011-2-15: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Sample code''' | | align="center" style="background:#f0f0f0;"|'''Sample code''' | ||
| align="center" style="background:#f0f0f0;"|'''Sex''' | | align="center" style="background:#f0f0f0;"|'''Sex''' | ||
| align="center" style="background:#f0f0f0;"|'''age (year.day)''' | |||
| align="center" style="background:#f0f0f0;"|'''Postmortem time''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ug/ul)/ volume (ul)''' | |||
|- | |- | ||
| 1 | | 1||5028||P1C||M||67.293||P18||0.5ug/ul/ 4ul||1947||P1P||M||70.251||P17||0.5ug/ul/ 4ul | ||
|- | |- | ||
| 2 | | 2||4789||P2C||F||72.053||P19||0.5ug/ul/ 4ul||4977||P2P||F||76.081||P14||0.5ug/ul/ 4ul | ||
|- | |- | ||
| 3 | | 3||5171||P3C||M||79.088||P05||0.5ug/ul/ 4ul||4879||P3P||M||75.351||P15||0.5ug/ul/ 4ul | ||
|- | |- | ||
| 4 | | 4||1818||P4C||M||76.294||P03||0.5ug/ul/ 4ul||4526||P4P||M||78.182||P01||0.5ug/ul/ 4ul | ||
|- | |- | ||
| 5 | | 5||5089||P5C||M||89.018||P14||0.5ug/ul/ 4ul||5203||P5P||M||89.06||P10||0.5ug/ul/ 4ul | ||
|- | |- | ||
| 6 | | 6||5237||P6C||M||52.291||P13||0.5ug/ul/ 4ul||1910||P6P||M||51.243||P10||0.5ug/ul/ 4ul | ||
|- | |- | ||
| 7 | | 7||1569||P7C1||F||77.089||P08||0.5ug/ul/ 4ul||1401||P7P||F||80.215||P04||0.5ug/ul/ 4ul | ||
|- | |- | ||
| 7 | | 7||5219||P7C2||F||76.348||P03||0.5ug/ul/ 4ul|||||||||||| | ||
|- | |- | ||
| 8 | | 8||4735||P8C||M||73.184||P21||0.5ug/ul/ 4ul||1741||P8P1||M||71.348||P20||0.5ug/ul/ 4ul | ||
|- | |- | ||
| 8 | | 8||||||||||||||5306||P8P2||M||76.311||P21||0.5ug/ul/ 4ul | ||
|}<br> | |}<br> | ||
Line 423: | Line 426: | ||
* so for lower amount of MmeI digested DNA, dilute adaptor with different dilution | * so for lower amount of MmeI digested DNA, dilute adaptor with different dilution | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''final conc''' | |||
| align="center" style="background:#f0f0f0;"|'''so for lower amount of MmeI digested DNA, dilute adaptor with different dilution''' | |||
|- | |||
| 4uM||20ng DNA, dilute 5x (2ul of 20uM adaptors with 8ul 1X Stoffel buffer) | |||
|- | |||
| 8uM||40ng DNA dilute 2.5x (4ul of 20uM adaptors with 6ul 1xStoffel buffer) | |||
|- | |||
| 12uM||60ng DNA dilute 1.67x (3ul of 20uM adaptors with 2ul 1x Stoffel buffer) | |||
|}<br> | |||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Group (by DNA amount)''' | | align="center" style="background:#f0f0f0;"|'''Group (by DNA amount)''' | ||
Line 496: | Line 509: | ||
**Gr. B = P4C | **Gr. B = P4C | ||
**Gr. C = P8P2 | **Gr. C = P8P2 | ||
* From PGP amplification, high percentage of N2Ligated | * From PGP amplification, high percentage of N2Ligated DNA template didn't show a good yield. I will reduce the amount of template to 1%, 2% and 5% and also reduce amount of primers to 1ul of 10uM in total volume 100ul (this exp do 50ul then just in proportion). | ||
* Note: this volume based on 100ng of MmeI digested DNA ligated to N2 adapters and purify with Ampure beads eluted volume 30ul. | * Note: this volume based on 100ng of MmeI digested DNA ligated to N2 adapters and purify with Ampure beads eluted volume 30ul. | ||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
Line 541: | Line 554: | ||
98C 30sec | 98C 30sec | ||
(98C 10sec -> | (98C 10sec -> 63C 20sec-> 72C 30sec) x 12 cycles | ||
72C 2min | 72C 2min | ||
15C hold.<br> | 15C hold.<br> | ||
- 2ul of PCR products were analyzed in PAGE gel<br> | - 2ul of PCR products were analyzed in PAGE gel<br> | ||
[[File:ZhangLab_2 2011-02-24 21hr 26min_PD N2Amp vary volume-1.jpg|400px]] [[File:ZhangLab_2 2011-02-24 21hr 26min_PD N2Amp vary volume-2.jpg|180px]] | |||
[[File:ZhangLab_2 2011-02-24 21hr 26min_PD N2Amp vary volume-1.jpg|400px]] [[File:ZhangLab_2 2011-02-24 21hr 26min_PD N2Amp vary volume-2.jpg|180px]]<br> | |||
* so from the gel above 2% of N2 ligated template will be used for library construction<br> | |||
* from the image labeling above, P8C2 should be P8P2 | |||
= Amplification = | |||
* Use 2% N2 ligated DNA template | |||
* 2 tubes/sample, total = 18*2 =36 plus 1NTC = 37<br> | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Reagents''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''38 rxn mix''' | |||
|- | |||
| N2 adapter ligated template||2.00||0.00 | |||
|- | |||
| 10uM PCR_F||1.00||38.00 | |||
|- | |||
| 10uM PCR_R.N2 IndX(X= 1, 2, 3,..,18)||1.00||0.00 | |||
|- | |||
| 50x SYBG||0.40||15.20 | |||
|- | |||
| 2x PhusionHF MM||50.00||1900.00 | |||
|- | |||
| H2O||45.60||1732.80 | |||
|- | |||
| Total volume (ul)||100.00||aliquot 97ul | |||
|- | |||
| ||||add 1ul of PCR_R IndX primer | |||
|- | |||
| ||||add 2ul of N2 ligated templated | |||
|}<br> | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Sample IDs''' | |||
| align="center" style="background:#f0f0f0;"|'''IndX''' | |||
|- | |||
| P1C||PCR_R.N2Ind1 | |||
|- | |||
| P2C||PCR_R.N2Ind2 | |||
|- | |||
| P3C||PCR_R.N2Ind3 | |||
|- | |||
| P4C||PCR_R.N2Ind4 | |||
|- | |||
| P5C||PCR_R.N2Ind5 | |||
|- | |||
| P6C||PCR_R.N2Ind6 | |||
|- | |||
| P7C1||PCR_R.N2Ind7 | |||
|- | |||
| P7C2||PCR_R.N2Ind8 | |||
|- | |||
| P8C||PCR_R.N2Ind9 | |||
|- | |||
| P1P||PCR_R.N2Ind10 | |||
|- | |||
| P2P||PCR_R.N2Ind11 | |||
|- | |||
| P3P||PCR_R.N2Ind12 | |||
|- | |||
| P4P||PCR_R.N2Ind13 | |||
|- | |||
| P5P||PCR_R.N2Ind14 | |||
|- | |||
| P6P||PCR_R.N2Ind15 | |||
|- | |||
| P7P||PCR_R.N2Ind16 | |||
|- | |||
| P8P1||PCR_R.N2Ind17 | |||
|- | |||
| P8P2||PCR_R.N2Ind18 | |||
|- | |||
| NTC||PCR_R.N2Ind18 | |||
|}<br> | |||
'''Program''' | |||
98C 30sec | |||
(98C 10sec -> 63C 20sec-> 72C 30sec) x 11 cycles | |||
72C 2min | |||
15C hold.<br> | |||
* Purify amplicon with 0.7volume Ampure beads, elute with 50ul EB | |||
* PAGE quantify DNA concentration (loaded sample 1ul) | |||
[[File:ZhangLab_2 2011-03-01 23hr 46min_PQ PC SeqLib.jpg|350px]] [[File:ZhangLab_2 2011-03-01 23hr 54min_PD patient SeqLib.jpg|350px]]<br> | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Sample ID''' | |||
| align="center" style="background:#f0f0f0;"|'''IndX''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc.(ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Total volume(ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 75ng (ul)''' | |||
|- | |||
| P1C||PCR_R.N2Ind1||4.61||50.00||230.53||16.27 | |||
|- | |||
| P2C||PCR_R.N2Ind2||11.84||50.00||592.24||6.33 | |||
|- | |||
| P3C||PCR_R.N2Ind3||7.48||50.00||374.09||10.02 | |||
|- | |||
| P4C||PCR_R.N2Ind4||7.88||50.00||393.95||9.52 | |||
|- | |||
| P5C||PCR_R.N2Ind5||9.12||50.00||456.20||8.22 | |||
|- | |||
| P6C||PCR_R.N2Ind6||8.09||50.00||404.66||9.27 | |||
|- | |||
| P7C1||PCR_R.N2Ind7||9.31||50.00||465.28||8.06 | |||
|- | |||
| P7C2||PCR_R.N2Ind8||3.75||50.00||187.49||20.00 | |||
|- | |||
| P8C||PCR_R.N2Ind9||3.32||50.00||165.96||22.60 | |||
|- | |||
| P1P||PCR_R.N2Ind10||3.86||50.00||192.75||19.45 | |||
|- | |||
| P2P||PCR_R.N2Ind11||5.70||50.00||285.03||13.16 | |||
|- | |||
| P3P||PCR_R.N2Ind12||2.74||50.00||137.01||27.37 | |||
|- | |||
| P4P||PCR_R.N2Ind13||10.63||50.00||531.64||7.05 | |||
|- | |||
| P5P||PCR_R.N2Ind14||5.14||50.00||257.20||14.58 | |||
|- | |||
| P6P||PCR_R.N2Ind15||3.64||50.00||182.19||20.58 | |||
|- | |||
| P7P||PCR_R.N2Ind16||6.07||50.00||303.29||12.36 | |||
|- | |||
| P8P1||PCR_R.N2Ind17||6.08||50.00||303.85||12.34 | |||
|- | |||
| P8P2||PCR_R.N2Ind18||12.84||50.00||642.25||5.84 | |||
|}<br> | |||
* perform PAGE size selection with 6% TBE 5-well gel (3gels) | |||
[[File:ZhangLab_2 2011-03-02 11hr 17min_PAGE SS SeqLib UPennPD PGP.jpg| 470px]] [[File:ZhangLab_2 2011-03-02 06hr 39min_PD SeqLib-2.jpg| 250px]] | |||
* Total volume of of multiplexed libraries: 60ul | |||
[[File:ZhangLab_2 2011-03-02 11hr 17min_PAGE SS SeqLib PD .jpg|400px]] | |||
*0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 10.2ng/ul, 49.9nM (336bp), yields = 10.2ng/ul * 60ul = 612ng | |||
* Sequencing: '''Sample ID: NP-220K-Parkinson_Ind1-18-Feb15, HL089 run''' | |||
* Data analysis: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-3-17]]<br> |
Latest revision as of 21:12, 6 September 2011
DMR220k capture(Probe set S1-S5 plus new probe set Exp1-Exp3) and library construction of Parkinson's patients[edit]
- Received 18 human brain samples from Pakinson's patients. This includes control and Pakinson's patiants (2011_02_02)
- Human frozen cerebral cortex
Pair | Control Sample ID | Sample code | Sex | age (year.day) | Postmortem time | Conc. (ug/ul)/ volume (ul) | PD Sample ID | Sample code | Sex | age (year.day) | Postmortem time | Conc. (ug/ul)/ volume (ul) |
1 | 5028 | P1C | M | 67.293 | P18 | 0.5ug/ul/ 4ul | 1947 | P1P | M | 70.251 | P17 | 0.5ug/ul/ 4ul |
2 | 4789 | P2C | F | 72.053 | P19 | 0.5ug/ul/ 4ul | 4977 | P2P | F | 76.081 | P14 | 0.5ug/ul/ 4ul |
3 | 5171 | P3C | M | 79.088 | P05 | 0.5ug/ul/ 4ul | 4879 | P3P | M | 75.351 | P15 | 0.5ug/ul/ 4ul |
4 | 1818 | P4C | M | 76.294 | P03 | 0.5ug/ul/ 4ul | 4526 | P4P | M | 78.182 | P01 | 0.5ug/ul/ 4ul |
5 | 5089 | P5C | M | 89.018 | P14 | 0.5ug/ul/ 4ul | 5203 | P5P | M | 89.06 | P10 | 0.5ug/ul/ 4ul |
6 | 5237 | P6C | M | 52.291 | P13 | 0.5ug/ul/ 4ul | 1910 | P6P | M | 51.243 | P10 | 0.5ug/ul/ 4ul |
7 | 1569 | P7C1 | F | 77.089 | P08 | 0.5ug/ul/ 4ul | 1401 | P7P | F | 80.215 | P04 | 0.5ug/ul/ 4ul |
7 | 5219 | P7C2 | F | 76.348 | P03 | 0.5ug/ul/ 4ul | ||||||
8 | 4735 | P8C | M | 73.184 | P21 | 0.5ug/ul/ 4ul | 1741 | P8P1 | M | 71.348 | P20 | 0.5ug/ul/ 4ul |
8 | 5306 | P8P2 | M | 76.311 | P21 | 0.5ug/ul/ 4ul |
Bisulfite conversion[edit]
- gDNA total volume 4ul (0.5ng/ul) so total amount = 2ug
- Add EB 6 ul to make volume to 10ul, so the final concentration should be 200ng/ul
Sample ID | Conc.(ng/ul) | Volume (ul) | H20 (ul) | CT conversion reagent (ul) | Total volume (ul) |
P1C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P2C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P3C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P4C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P5C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P6C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P7C1 | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P7C2 | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P8C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P1P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P2P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P3P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P4P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P5P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P6P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P7P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P8P1 | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P8P2 | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
- follow standard protocol for bisulfite conversion of ZymoResearch
- elute with 17ul Elution buffer
Determine bisulfite converted gDNA concentration using Qubit Fluorometer[edit]
Sample preparation
- Add 1ul of BIS-CVT gDNA to 199ul of Quant-iT working solution
Sample ID | Concentration in the Qubit | ' | uL used | Dilution | Sample Concentration | ' | Final conc.(ng/ul) | Yields (ng) |
P1C | 150.00 | ng/mL | 1.00 | 200.00 | 150.00 | ng/mL | 30.00 | 510.00 |
P2C | 118.00 | ng/mL | 1.00 | 200.00 | 118.00 | ng/mL | 23.60 | 401.20 |
P3C | 59.40 | ng/mL | 1.00 | 200.00 | 59.40 | ng/mL | 11.88 | 201.96 |
P4C | 321.00 | ng/mL | 1.00 | 200.00 | 321.00 | ng/mL | 64.20 | 1091.40 |
P5C | 34.70 | ng/mL | 1.00 | 200.00 | 34.70 | ng/mL | 6.94 | 117.98 |
P6C | 29.10 | ng/mL | 1.00 | 200.00 | 29.10 | ng/mL | 5.82 | 98.94 |
P7C1 | 33.70 | ng/mL | 1.00 | 200.00 | 33.70 | ng/mL | 6.74 | 114.58 |
P7C2 | 29.60 | ng/mL | 1.00 | 200.00 | 29.60 | ng/mL | 5.92 | 100.64 |
P8C | 57.60 | ng/mL | 1.00 | 200.00 | 57.60 | ng/mL | 11.52 | 195.84 |
P1P | 189.00 | ng/mL | 1.00 | 200.00 | 189.00 | ng/mL | 37.80 | 642.60 |
P2P | 393.00 | ng/mL | 1.00 | 200.00 | 393.00 | ng/mL | 78.60 | 1336.20 |
P3P | 337.00 | ng/mL | 1.00 | 200.00 | 337.00 | ng/mL | 67.40 | 1145.80 |
P4P | 307.00 | ng/mL | 1.00 | 200.00 | 307.00 | ng/mL | 61.40 | 1043.80 |
P5P | 19.10 | ng/mL | 1.00 | 200.00 | 19.10 | ng/mL | 3.82 | 64.94 |
P6P | 48.00 | ng/mL | 1.00 | 200.00 | 48.00 | ng/mL | 9.60 | 163.20 |
P7P | 18.90 | ng/mL | 1.00 | 200.00 | 18.90 | ng/mL | 3.78 | 64.26 |
P8P1 | 44.60 | ng/mL | 1.00 | 200.00 | 44.60 | ng/mL | 8.92 | 151.64 |
P8P2 | 37.80 | ng/mL | 1.00 | 200.00 | 37.80 | ng/mL | 7.56 | 128.52 |
Capture set up[edit]
Probe calculation[edit]
Probe/target ratio | 100 | ' |
Probe size (sub set Exp1-3) | 55,000 each | |
Template | 200ng | |
Human gDNA MW | 1.82E+12g/mol | (3E9bp x 607.4D/bp + 157) |
Human gDNA 200ng | 1.10E-19mol | |
Probe required (100:1) | 1.10E-17 mol | |
Probe MW (each set) | 1.72E9 g/mol | (55,000 x 103bp x 303.7D/bp + 79D) |
Amount probe required | 18.90ng each |
- Note: for DMR220k subset S1-S5 used Multiplier C
Subset ID | Multiplier C | Amount probes required x multiplier (ng) | Conc(ng/ul) | Volume (ul) | 20 rxn |
DMR.s1 | 0.05 | 0.11 | 2.00 | 0.06 | 1.10 |
DMR.s2 | 0.09 | 0.21 | 2.00 | 0.11 | 2.10 |
DMR.s3 | 0.25 | 4.71 | 23.20 | 0.20 | 4.06 |
DMR.s4 | 1.00 | 18.88 | 24.80 | 0.76 | 15.23 |
DMR.s5 | 4.83 | 91.22 | 24.70 | 3.69 | 73.86 |
DMR.Exp1 | 1.00 | 18.90 | 28.00 | 0.68 | 13.50 |
DMR.Exp2 | 1.00 | 18.90 | 26.00 | 0.73 | 14.54 |
DMR.Exp3 | 1.00 | 18.90 | 25.00 | 0.76 | 15.12 |
Total volume (ul) | 6.98 | 139.51 |
- Note: used probe set DMR220k S5, Exp1-3 prepared on 2011_02_02
Reaction mix[edit]
Components | 1rxn (ul) | 20rxn mix (ul) |
10X AmpLigase Buffer | 2.55 | 51.00 |
DMR220k Exp1-3 probe mix (ul) | 6.98 | 139.60 |
105nM Oligo suppressor | 1.00 | 20.00 |
Total volume (ul) | 10.53 | 210.60 |
Sample ID | Final conc.(ng/ul) | volume for 200ng | H2O | probe mix (ul) | 10x AmpLigase buffer (ul) | 105nM Oligo suppressor (ul) | Total volume (ul) | Total amount of bis-cvt DNA captured |
P1C | 30.00 | 6.67 | 8.33 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P2C | 23.60 | 8.47 | 6.53 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P3C* | 11.88 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 178.20 |
P4C | 64.20 | 3.12 | 11.88 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P5C* | 6.94 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 104.10 |
P6C* | 5.82 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 87.30 |
P7C1* | 6.74 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 101.10 |
P7C2* | 5.92 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 88.80 |
P8C* | 11.52 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 172.80 |
P1P | 37.80 | 5.29 | 9.71 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P2P | 78.60 | 2.54 | 12.46 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P3P | 67.40 | 2.97 | 12.03 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P4P | 61.40 | 3.26 | 11.74 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P5P* | 3.82 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 57.30 |
P6P* | 9.60 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 144.00 |
P7P* | 3.78 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 56.70 |
P8P1* | 8.92 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 133.80 |
P8P2* | 7.56 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 113.40 |
- Note: * less than 200ng of bis-cvt DNA in probe capture
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) (2011_02_16) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
PCR Amplification[edit]
(2011_02_17)
- amplify 2tubes/ sample
- 19samples x 2 = 38 plus 1 NTC = 39rxn
Reagents | volume (ul) | 39.5rxn mix |
captured template | 9.00 | 0.00 |
100uM AmpFV6.3NH2 | 0.20 | 7.90 |
100uM AmpRV6.3NH2 | 0.20 | 7.90 |
50x SYBG | 0.40 | 15.80 |
2x PhusionHF MM | 50.00 | 1975.00 |
H2O | 40.20 | 1587.90 |
Total volume (ul) | 100.00 | 3594.50 |
aliquot 91ul, add 9ul of captured DNA template
Program
98C 30sec (98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles 72C 2min 15C hold. - purify with 0.7vol. Ampure beads, elute with 25ul EB/tube, so total volume = 75ul - PAGE quantify DNA concentration (loaded sample 1ul)
File:ZhangLab 2 2011-02-22 15hr 46min 1st Amp P1C-P8C.jpg File:ZhangLab 2 2011-02-22 15hr 46min 1st Amp P1P-P2P2.jpg
PAGE quantification and MmeI digestion set up[edit]
Sample ID | Conc.(ng/ul) | Total volume(ul) | Total amount (ng) | 10X NEB buffer4 | 32mM SAM | MmeI | Total volume(ul) |
P1C | 2.09 | 75.00 | 156.39 | 9.30 | 0.30 | 8.00 | 92.60 |
P2C | 4.67 | 75.00 | 350.41 | 9.30 | 0.30 | 8.00 | 92.60 |
P3C | 3.09 | 75.00 | 231.84 | 9.30 | 0.30 | 8.00 | 92.60 |
P4C | 4.24 | 75.00 | 317.74 | 9.30 | 0.30 | 8.00 | 92.60 |
P5C | 3.45 | 75.00 | 258.94 | 9.30 | 0.30 | 8.00 | 92.60 |
P6C | 4.19 | 75.00 | 314.13 | 9.30 | 0.30 | 8.00 | 92.60 |
P7C1 | 3.74 | 75.00 | 280.28 | 9.30 | 0.30 | 8.00 | 92.60 |
P7C2 | 2.14 | 75.00 | 160.76 | 9.30 | 0.30 | 8.00 | 92.60 |
P8C | 3.21 | 75.00 | 240.62 | 9.30 | 0.30 | 8.00 | 92.60 |
P1P | 3.35 | 75.00 | 251.00 | 9.30 | 0.30 | 8.00 | 92.60 |
P2P | 3.18 | 75.00 | 238.35 | 9.30 | 0.30 | 8.00 | 92.60 |
P3P | 3.01 | 75.00 | 225.81 | 9.30 | 0.30 | 8.00 | 92.60 |
P4P | 5.10 | 75.00 | 382.61 | 9.30 | 0.30 | 8.00 | 92.60 |
P5P | 4.06 | 75.00 | 304.36 | 9.30 | 0.30 | 8.00 | 92.60 |
P6P | 3.22 | 75.00 | 241.75 | 9.30 | 0.30 | 8.00 | 92.60 |
P7P | 3.68 | 75.00 | 276.35 | 9.30 | 0.30 | 8.00 | 92.60 |
P8P1 | 4.03 | 75.00 | 302.36 | 9.30 | 0.30 | 8.00 | 92.60 |
P8P2 | 7.34 | 75.00 | 550.50 | 9.30 | 0.30 | 8.00 | 92.60 |
Reagents | 1x | 18.5 rxn mix |
10X NEB buffer4 | 9.30 | 172.05 |
32mM SAM | 0.30 | 5.55 |
MmeI | 8.00 | 148.00 |
Total | 17.60 | aliquot 17.6ul, add 75ul of amplified amplicon |
- incubate 37C 2hr
- purify with 0.7vol. Ampure beads, elute with 30ul EB
- PAGE quantify DNA concentration (loaded sample 2ul)
File:ZhangLab 2 2011-02-22 20hr 38min MmeI digestion P1C-P8C.jpg File:ZhangLab 2 2011-02-22 20hr 41min MmeI digestion P1P-P8P2.jpg
Sample ID | Conc of MmeI completely digested DNA (ng/ul) | Total volume (ul) | Total amount (ng) |
P1C | 0.56 | 30.00 | 16.84 |
P2C | 1.60 | 30.00 | 47.94 |
P3C | 0.97 | 30.00 | 28.97 |
P4C | 1.33 | 30.00 | 39.91 |
P5C | 1.42 | 30.00 | 42.46 |
P6C | 0.99 | 30.00 | 29.61 |
P7C1 | 1.41 | 30.00 | 42.35 |
P7C2 | 0.46 | 30.00 | 13.71 |
P8C | 0.57 | 30.00 | 17.01 |
P1P | 0.59 | 30.00 | 17.62 |
P2P | 0.94 | 30.00 | 28.29 |
P3P | 0.61 | 30.00 | 18.17 |
P4P | 1.59 | 30.00 | 47.80 |
P5P | 0.92 | 30.00 | 27.58 |
P6P | 0.42 | 30.00 | 12.64 |
P7P | 1.15 | 30.00 | 34.45 |
P8P1 | 1.29 | 30.00 | 38.72 |
P8P2 | 2.03 | 30.00 | 60.95 |
N2 adaptor ligation set up[edit]
- group sample into 3 different groups based on total completely MmeI digested DNA amount including
- groupA: <= 20ng
- 20ng < groupB <= 40ng
- 40ng < groupC <= 60ng
Adaptor preparation[edit]
Components | Volume (ul) | Final conc. (uM) |
100uM PE_N2_adaptor | 2.00 | 20.00 |
100uM PE_b_A | 2.00 | 20.00 |
10x Stoffel buffer | 1.00 | 1x |
H2O | 5.00 | |
Total | 10.00 |
Program
94C 2min -> 0.2C/sec to 20C -> 4C hold
Adapters to ligated product ratio: 10:1
Est length of digested products: 212bp (after MmeI)
MW digested products = (212bp*607.4 D/bp +157.9 D) = 128.927kD = 128,927 g/mole
For 100 ng digested product = 100ng / 128,927g/mole = 7.76E-4 nmole * 10:1 = 0.008 nmole adapters required.
20uM Adapters (ul) = 0.008nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.008nmoles/ (20E-3 nmoles/ul) = 0.40 ul
- 0.40 ul of 20uM adapters per 100ng digested products.
- so for lower amount of MmeI digested DNA, dilute adaptor with different dilution
final conc | so for lower amount of MmeI digested DNA, dilute adaptor with different dilution |
4uM | 20ng DNA, dilute 5x (2ul of 20uM adaptors with 8ul 1X Stoffel buffer) |
8uM | 40ng DNA dilute 2.5x (4ul of 20uM adaptors with 6ul 1xStoffel buffer) |
12uM | 60ng DNA dilute 1.67x (3ul of 20uM adaptors with 2ul 1x Stoffel buffer) |
Group (by DNA amount) | Samples | conc.(ng/ul) | Volume (ul) | Total amount (ng) | N2 adapters conc (uM) | N2 adaptors volume (ul) | 5xQuickLigase buffer | QuickLigase (ul) | H2O (ul) | Total (ul) |
A | P1C | 0.56 | 30.00 | 16.84 | 4.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
C | P2C | 1.60 | 30.00 | 47.94 | 12.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P3C | 0.97 | 30.00 | 28.97 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P4C | 1.33 | 30.00 | 39.91 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P5C | 1.42 | 30.00 | 42.46 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P6C | 0.99 | 30.00 | 29.61 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P7C1 | 1.41 | 30.00 | 42.35 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
A | P7C2 | 0.46 | 30.00 | 13.71 | 4.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
A | P8C | 0.57 | 30.00 | 17.01 | 4.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
A | P1P | 0.59 | 30.00 | 17.62 | 4.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P2P | 0.94 | 30.00 | 28.29 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
A | P3P | 0.61 | 30.00 | 18.17 | 4.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
C | P4P | 1.59 | 30.00 | 47.80 | 12.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P5P | 0.92 | 30.00 | 27.58 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
A | P6P | 0.42 | 30.00 | 12.64 | 4.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P7P | 1.15 | 30.00 | 34.45 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
B | P8P1 | 1.29 | 30.00 | 38.72 | 8.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
C | P8P2 | 2.03 | 30.00 | 60.95 | 12.00 | 0.40 | 8.00 | 1.00 | 0.60 | 39.40 |
Reagents | 1x | Mix A (6.5 rxn mix) | Mix B (9.5 rxn mix) | Mix C (4.5 rxn mix) |
5xQuickLigase buffer | 8.00 | 52.00 | 76.00 | 36.00 |
QuickLigase | 1.00 | 6.50 | 9.50 | 4.50 |
N2 Adapters, conc. (Gr. A, B or C) | 0.40 | 2.60 | 3.80 | 1.80 |
H2O | 0.60 | 3.90 | 5.70 | 2.70 |
Total | 10.00 | 65.00 | 95.00 | 45.00 |
- incubate at RT for 15 min
- purify with Ampure beads, elute with 30ul EB
Amplification with N2 barcoded primers[edit]
- vary volume of DNA template 2%, 5% and 10% in different group A, B and C
- Gr. A = P1C
- Gr. B = P4C
- Gr. C = P8P2
- From PGP amplification, high percentage of N2Ligated DNA template didn't show a good yield. I will reduce the amount of template to 1%, 2% and 5% and also reduce amount of primers to 1ul of 10uM in total volume 100ul (this exp do 50ul then just in proportion).
- Note: this volume based on 100ng of MmeI digested DNA ligated to N2 adapters and purify with Ampure beads eluted volume 30ul.
Reagents | 1% template | 4.2rxn mix | 2% template | 4.2rxn mix | 5% template | 4.2rxn mix |
N2 adapter ligated template | 0.50 | 0.00 | 1.00 | 0.00 | 2.50 | 0.00 |
10uM PCR_F | 0.50 | 2.10 | 0.50 | 2.10 | 0.50 | 2.10 |
10uM PCR_R.N2 IndX(X= 1, 2 and 4) | 0.50 | 0.00 | 0.50 | 0.00 | 0.50 | 0.00 |
50x SYBG | 0.20 | 0.84 | 0.20 | 0.84 | 0.20 | 0.84 |
2x PhusionHF MM | 25.00 | 105.00 | 25.00 | 105.00 | 25.00 | 105.00 |
H2O | 23.30 | 97.86 | 22.80 | 95.76 | 21.30 | 89.46 |
Total volume (ul) | 50.00 | aliquot 49ul | 50.00 | aliquot 48.5ul | 50.00 | aliquot 47ul |
add 0.5ul template | add 1ul template | add 2.5ul template | ||||
add 0.5ul R primer | add 0.5ul R primer | add 0.5ul R primer |
Sample IDs | IndX |
P1C | PCR_R.N2Ind1 |
P2C | PCR_R.N2Ind2 |
P8P2 | PCR_R.N2Ind18 |
NTC | PCR_R.N2Ind18 |
Program
98C 30sec (98C 10sec -> 63C 20sec-> 72C 30sec) x 12 cycles 72C 2min 15C hold.
- 2ul of PCR products were analyzed in PAGE gel
File:ZhangLab 2 2011-02-24 21hr 26min PD N2Amp vary volume-1.jpg File:ZhangLab 2 2011-02-24 21hr 26min PD N2Amp vary volume-2.jpg
- so from the gel above 2% of N2 ligated template will be used for library construction
- from the image labeling above, P8C2 should be P8P2
Amplification[edit]
- Use 2% N2 ligated DNA template
- 2 tubes/sample, total = 18*2 =36 plus 1NTC = 37
Reagents | 1 rxn | 38 rxn mix |
N2 adapter ligated template | 2.00 | 0.00 |
10uM PCR_F | 1.00 | 38.00 |
10uM PCR_R.N2 IndX(X= 1, 2, 3,..,18) | 1.00 | 0.00 |
50x SYBG | 0.40 | 15.20 |
2x PhusionHF MM | 50.00 | 1900.00 |
H2O | 45.60 | 1732.80 |
Total volume (ul) | 100.00 | aliquot 97ul |
add 1ul of PCR_R IndX primer | ||
add 2ul of N2 ligated templated |
Sample IDs | IndX |
P1C | PCR_R.N2Ind1 |
P2C | PCR_R.N2Ind2 |
P3C | PCR_R.N2Ind3 |
P4C | PCR_R.N2Ind4 |
P5C | PCR_R.N2Ind5 |
P6C | PCR_R.N2Ind6 |
P7C1 | PCR_R.N2Ind7 |
P7C2 | PCR_R.N2Ind8 |
P8C | PCR_R.N2Ind9 |
P1P | PCR_R.N2Ind10 |
P2P | PCR_R.N2Ind11 |
P3P | PCR_R.N2Ind12 |
P4P | PCR_R.N2Ind13 |
P5P | PCR_R.N2Ind14 |
P6P | PCR_R.N2Ind15 |
P7P | PCR_R.N2Ind16 |
P8P1 | PCR_R.N2Ind17 |
P8P2 | PCR_R.N2Ind18 |
NTC | PCR_R.N2Ind18 |
Program
98C 30sec (98C 10sec -> 63C 20sec-> 72C 30sec) x 11 cycles 72C 2min 15C hold.
- Purify amplicon with 0.7volume Ampure beads, elute with 50ul EB
- PAGE quantify DNA concentration (loaded sample 1ul)
File:ZhangLab 2 2011-03-01 23hr 46min PQ PC SeqLib.jpg File:ZhangLab 2 2011-03-01 23hr 54min PD patient SeqLib.jpg
Sample ID | IndX | Conc.(ng/ul) | Total volume(ul) | Total amount (ng) | Volume for 75ng (ul) |
P1C | PCR_R.N2Ind1 | 4.61 | 50.00 | 230.53 | 16.27 |
P2C | PCR_R.N2Ind2 | 11.84 | 50.00 | 592.24 | 6.33 |
P3C | PCR_R.N2Ind3 | 7.48 | 50.00 | 374.09 | 10.02 |
P4C | PCR_R.N2Ind4 | 7.88 | 50.00 | 393.95 | 9.52 |
P5C | PCR_R.N2Ind5 | 9.12 | 50.00 | 456.20 | 8.22 |
P6C | PCR_R.N2Ind6 | 8.09 | 50.00 | 404.66 | 9.27 |
P7C1 | PCR_R.N2Ind7 | 9.31 | 50.00 | 465.28 | 8.06 |
P7C2 | PCR_R.N2Ind8 | 3.75 | 50.00 | 187.49 | 20.00 |
P8C | PCR_R.N2Ind9 | 3.32 | 50.00 | 165.96 | 22.60 |
P1P | PCR_R.N2Ind10 | 3.86 | 50.00 | 192.75 | 19.45 |
P2P | PCR_R.N2Ind11 | 5.70 | 50.00 | 285.03 | 13.16 |
P3P | PCR_R.N2Ind12 | 2.74 | 50.00 | 137.01 | 27.37 |
P4P | PCR_R.N2Ind13 | 10.63 | 50.00 | 531.64 | 7.05 |
P5P | PCR_R.N2Ind14 | 5.14 | 50.00 | 257.20 | 14.58 |
P6P | PCR_R.N2Ind15 | 3.64 | 50.00 | 182.19 | 20.58 |
P7P | PCR_R.N2Ind16 | 6.07 | 50.00 | 303.29 | 12.36 |
P8P1 | PCR_R.N2Ind17 | 6.08 | 50.00 | 303.85 | 12.34 |
P8P2 | PCR_R.N2Ind18 | 12.84 | 50.00 | 642.25 | 5.84 |
- perform PAGE size selection with 6% TBE 5-well gel (3gels)
File:ZhangLab 2 2011-03-02 11hr 17min PAGE SS SeqLib UPennPD PGP.jpg File:ZhangLab 2 2011-03-02 06hr 39min PD SeqLib-2.jpg
- Total volume of of multiplexed libraries: 60ul
File:ZhangLab 2 2011-03-02 11hr 17min PAGE SS SeqLib PD .jpg
- 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 10.2ng/ul, 49.9nM (336bp), yields = 10.2ng/ul * 60ul = 612ng
- Sequencing: Sample ID: NP-220K-Parkinson_Ind1-18-Feb15, HL089 run
- Data analysis: [[1]]