Noi/NOTES/2011-2-15: Difference between revisions
>Noi No edit summary |
>Noi No edit summary |
||
Line 501: | Line 501: | ||
| align="center" style="background:#f0f0f0;"|'''4.2rxn mix''' | | align="center" style="background:#f0f0f0;"|'''4.2rxn mix''' | ||
| align="center" style="background:#f0f0f0;"|'''5% template''' | | align="center" style="background:#f0f0f0;"|'''5% template''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''4.2rxn mix''' | ||
| align="center" style="background:#f0f0f0;"|'''10% template''' | | align="center" style="background:#f0f0f0;"|'''10% template''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''4.2rxn mix''' | ||
|- | |- | ||
| N2 adapter ligated template||1.00||0.00||2.50||0.00||5.00||0.00 | | N2 adapter ligated template||1.00||0.00||2.50||0.00||5.00||0.00 |
Revision as of 01:56, 24 February 2011
DMR220k capture(Probe set S1-S5 plus new probe set Exp1-Exp3) and library construction of Parkinson's patients
- 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 |
1. | 5028 | P1C | M | 67.29 | P18 | 0.5ug/ul/ 4ul | 1947 | P1P | M |
2. | 4789 | P2C | F | 72.05 | P19 | 0.5ug/ul/ 4ul | 4977 | P2P | F |
3. | 5171 | P3C | M | 79.09 | P05 | 0.5ug/ul/ 4ul | 4879 | P3P | M |
4. | 1818 | P4C | M | 76.29 | P03 | 0.5ug/ul/ 4ul | 4526 | P4P | M |
5. | 5089 | P5C | M | 89.02 | P14 | 0.5ug/ul/ 4ul | 5203 | P5P | M |
6. | 5237 | P6C | M | 52.29 | P13 | 0.5ug/ul/ 4ul | 1910 | P6P | M |
7. | 1569 | P7C1 | F | 77.09 | P08 | 0.5ug/ul/ 4ul | 1401 | P7P | F |
7. | 5219 | P7C2 | F | 76.35 | P03 | 0.5ug/ul/ 4ul | |||
8. | 4735 | P8C | M | 73.18 | P21 | 0.5ug/ul/ 4ul | 1741 | P8P1 | M |
8. | 5306 | P8P2 | M |
Bisulfite conversion
- 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
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
Probe calculation
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
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
(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
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
- 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
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
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
- 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
Reagents | 2% template | 4.2rxn mix | 5% template | 4.2rxn mix | 10% template | 4.2rxn mix |
N2 adapter ligated template | 1.00 | 0.00 | 2.50 | 0.00 | 5.00 | 0.00 |
10uM PCR_F | 1.00 | 4.20 | 1.00 | 4.20 | 1.00 | 4.20 |
10uM PCR_R.N2 IndX(X= 1, 2 and 4) | 1.00 | 0.00 | 1.00 | 0.00 | 1.00 | 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 | 21.80 | 91.56 | 20.30 | 85.26 | 17.80 | 74.76 |
Total volume (ul) | 50.00 | aliquot 48ul | 50.00 | aliquot 46.5ul | 50.00 | aliquot 44ul |
add 1ul template | add 2.5 template | add 5ul template | ||||
add 1ul R primer | add 1ul R primer | add 1ul 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 -> 62C 20sec-> 72C 30sec) x 12 cycles 72C 2min 15C hold.
- 2ul of PCR products were analyzed in PAGE gel