Noi/NOTES/2013-12-24: Difference between revisions
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>Noi |
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:- Loaded XX ul of each sample | :- Loaded XX ul of each sample | ||
'''GP1V4'''<br> | '''GP1V4'''<br> | ||
[[File:ZhangLab_2 2013-12-25 19hr 44min_GP1_V4_1-8_PQ.jpg| 400px]] | [[File:ZhangLab_2 2013-12-25 19hr 44min_GP1_V4_1-8_PQ.jpg| 400px]]<br> | ||
'''GP1V6'''<br> | '''GP1V6'''<br> | ||
[[File:ZhangLab_2 2013-12-25 19hr 46min_GP1_V6_1-8_PQ.jpg|400px]] | [[File:ZhangLab_2 2013-12-25 19hr 46min_GP1_V6_1-8_PQ.jpg|400px]] | ||
==== PAGE size-selection ==== | ==== PAGE size-selection ==== | ||
[[File:ZhangLab_2 2013-12-26 13hr 29min_PAGE-SS_V4.jpg|200px]] [[File:ZhangLab_2 2013-12-26 13hr 42min_PAGE-SS_V6.jpg|200px]] | [[File:ZhangLab_2 2013-12-26 13hr 29min_PAGE-SS_V4.jpg|200px]] [[File:ZhangLab_2 2013-12-26 13hr 42min_PAGE-SS_V6.jpg|200px]] |
Revision as of 18:54, 17 May 2014
BSPP capture of GP1 samples
- I have helped Alan to continue GP1 BSPP preparation and BSPP capture on cancer cell and blood samples.
- Alan's note
- Alan performed 24 PCR reaction for production PCR for 15 cycles and I continued EtOH precipitation and column re-purification.
- EtOH precipitation of the 24 rxn PCR in2x 15mL tube
- 1200ul amplified amplicons + 120ul 3M NaOAc pH5.5 + 4ul Glycoblue and 3mL 100% EtOH
- Incubate at -80C ~5hr -->
- Resuspend each tube with 100ul H2O, purified with 2X QIAGEN column, eluted 50ul each tube. After ND measurement, total volume for each set is ~90ul)
- V4 set: conc. dsDNA (ND) = 54.7ng/ul, total yield = 90ul * 54.7ng/ul = 4.92ug
- V6 set: conc. dsDNA (ND) = 68.4ng/ul, total yield = 90ul * 68.4ng/ul = 6.16ug
- Note. The total yield was expected to be ~25+/-5ug for 24 reactions, but I got only XX ug. This is possible that, Alan amplified only 9 cycles in expansion PCR. Since >90% of seed oligos from CustomAraays are inclomplete oligoes. There could be more fraction of these incomplete oligos in the first amplicons as we used very high template concentration and amplified with fewer cycles (I always amplified 15 cycles for expansion PCR and production PCR consistently.)
Lambda Exonuclease digestion
Components | 1x rxn |
ds amplicons | 90.00 |
10X Lambda Exo buffer | 10.50 |
Lambda Exo (5U/ul) | 5.00 |
Total | 105.50 |
- Incubate at 37C for 1hr
- Purified with 1X QIAQuick column for each set (I added 7x vol of PB buffer = 770ul. Total volume is 875ul, so I add 750ul of solution mix -> spin and add the rest to the column again.)
- Eluted with 40ul EB buffer each (total volume left after ND measurement ~37.5ul)
- V4 set: conc. ssDNA (ND) = 34.6ng/ul, yield = 37.5ul * 34.6ng/ul = 1.3ug
- V6 set: conc. ssDNA (ND) = 49.7ng/ul, yield = 37.5ul * 49.7ng/ul = 1.86ug
- I will finish up probe prep later
BSPP capture
- Alan extracted gDNA of 5 cancer cell lines. He split the cell into duplicates, so there were two tubes of purified gDNA.
- He measured gDNA conc. of cancer cell line and blood sample by Qubit dsDNA assay kit.
Qubit result
Sample | Sample Concentration (ng/ul) |
BXPC3_1 | 63.00 |
BXPC3_2 | 70.40 |
U87MG_1 | 57.20 |
U87MG_2 | 63.00 |
PANC1_1 | 52.40 |
PANC1_2 | 57.80 |
BE(2)C_1 | 49.80 |
BE(2)C_2 | 50.00 |
T98G_1 | 68.20 |
T98G_2 | 69.20 |
B1 | 102.00 |
D1 | 110.00 |
F10 | 106.00 |
G10 | 108.00 |
G11 | 82.60 |
H11 | 102.00 |
Bisulfite conversion
Sample IDs and labeling matching
Sample IDs | V4 Labeled IDs | V6 labeled IDs |
BXPC3_1 | V4-1 | V6-1 |
U87MG_1 | V4-2 | V6-2 |
PANC1_1 | V4-3 | V6-3 |
BE(2)C_1 | V4-4 | V6-4 |
T98G_1 | V4-5 | V6-5 |
B1 | V4-6 | V6-6 |
D1 | V4-7 | V6-7 |
H11 | V4-8 | V6-8 |
- I used set _1 of cancel cells and B1, D1 and H11 blood samples for BSPP capture.
- I used the new DNA methylation lightning kit (column) ordered by Dr. Zhang in November 2013.
- I did bisulfite conversion in duplicates for the two probe sets capture.
Samples | Conc. (ng/ul) | Volume for 600ng (ul) | Go to tube | H2O | Lightning Conv Reagent | Total (ul) |
BXPC3_1 | 63.00 | 9.52 | A1 | 10.48 | 130.00 | 150.00 |
U87MG_1 | 57.20 | 10.49 | A2 | 9.51 | 130.00 | 150.00 |
PANC1_1 | 52.40 | 11.45 | A3 | 8.55 | 130.00 | 150.00 |
BE(2)C_1 | 49.80 | 12.05 | A4 | 7.95 | 130.00 | 150.00 |
T98G_1 | 68.20 | 8.80 | A5 | 11.20 | 130.00 | 150.00 |
B1 | 102.00 | 5.88 | A6 | 14.12 | 130.00 | 150.00 |
D1 | 110.00 | 5.45 | A7 | 14.55 | 130.00 | 150.00 |
H11 | 102.00 | 5.88 | A8 | 14.12 | 130.00 | 150.00 |
BXPC3_1 | 63.00 | 9.52 | B1 | 10.48 | 130.00 | 150.00 |
U87MG_1 | 57.20 | 10.49 | B2 | 9.51 | 130.00 | 150.00 |
PANC1_1 | 52.40 | 11.45 | B3 | 8.55 | 130.00 | 150.00 |
BE(2)C_1 | 49.80 | 12.05 | B4 | 7.95 | 130.00 | 150.00 |
T98G_1 | 68.20 | 8.80 | B5 | 11.20 | 130.00 | 150.00 |
B1 | 102.00 | 5.88 | B6 | 14.12 | 130.00 | 150.00 |
D1 | 110.00 | 5.45 | B7 | 14.55 | 130.00 | 150.00 |
H11 | 102.00 | 5.88 | B8 | 14.12 | 130.00 | 150.00 |
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. The volume left after assay ~16ul
ssDNA Qubit assay result
Sample | Concentration in the Qubit | ' | uL used | Dilution | Sample conc. (ng/ul) |
V4-1 | 63.7 | ng/mL | 1 | 200 | 12.7 |
V4-2 | 71.4 | ng/mL | 1 | 200 | 14.3 |
V4-3 | 51.4 | ng/mL | 1 | 200 | 10.3 |
V4-4 | 30.9 | ng/mL | 1 | 200 | 6.18 |
V4-5 | 57.9 | ng/mL | 1 | 200 | 11.6 |
V4-6 | 281 | ng/mL | 1 | 200 | 56.2 |
V4-7 | 70.1 | ng/mL | 1 | 200 | 14 |
V4-8 | 125 | ng/mL | 1 | 200 | 25 |
V6-1 | 85.1 | ng/mL | 1 | 200 | 17 |
V6-2 | 61.3 | ng/mL | 1 | 200 | 12.3 |
V6-3 | 61.3 | ng/mL | 1 | 200 | 12.3 |
V6-4 | 70.7 | ng/mL | 1 | 200 | 14.1 |
V6-5 | 77.1 | ng/mL | 1 | 200 | 15.4 |
V6-6 | 112 | ng/mL | 1 | 200 | 22.5 |
V6-7 | 35.6 | ng/mL | 1 | 200 | 7.12 |
V6-8 | 100 | ng/mL | 1 | 200 | 20 |
BSPP capture set up
Probe calculation
GP1_V4
Probe:taeget | 500:1 | Unit | Calculation |
Probe size (number of individual probe in oligo mix) | 7110 | probes | |
Human bis-cvt gDNA required | 200 | ng | |
Human gDNA MW | 1.822E+12 | g/mole | 3.0E+09 * 607.4Da/bp |
Human gDNA( 200ng) | 1.098E-19 | mole | 200E-09g / 1.822E+12g/mole |
Probe required (500:1) | 5.488E-17 | mole | 500 * 1.098E-19mole |
Probe mix MW (size 7110probes, 110nt) | 2.375E+08 | g/mole | 7110 probes * 110bp *303.7Da/bp |
Mass of probe required | 1.304E-08 | g | 5.448E-17 * 2.375E+08mole |
Mass of probe required | 13 | ng | |
Probe conc | 4.54 | ng/ul | |
Volume of probe | 2.86 | ul |
- Note: Probe is not enough, so use 2.5ul/sample||= 11.35||ng
GP1_V6
Probe:taeget | 500:1 | Unit | Calculation |
Probe size (number of individual probe in oligo mix) | 3662 | probes | |
Human bis-cvt gDNA required | 200 | ng | |
Human gDNA MW | 1.822E+12 | g/mole | 3.0E+09 * 607.4Da/bp |
Human gDNA( 200ng) | 1.098E-19 | mole | 200E-09g / 1.822E+12g/mole |
Probe required (500:1) | 5.488E-17 | mole | 500 * 1.098E-19mole |
Probe mix MW (size 7110probes, 110nt) | 1.223E+08 | g/mole | 3662 probes * 110bp *303.7Da/bp |
Mass of probe required | 6.714E-09 | g | 5.448E-17 * 1.223E+08mole |
Mass of probe required | 6.71 | ng | |
Probe conc | 7.96 | ng/ul | |
Volume of probe | 0.84 | ul |
GP1_V4 set up
Bis-cvt samples | Conc. (ng/ul) | Volume for 200ng (ul) | Bis-cvt samples | H2O (ul) | GP1_V4 | 10X AmpLigase Buffer | Total (ul) | Converted DNA amount |
V4-1 | 12.70 | 15.50 | V4-1 | 0.00 | 2.50 | 2.00 | 20.00 | 196.85 |
V4-2 | 14.30 | 13.99 | V4-2 | 1.51 | 2.50 | 2.00 | 20.00 | 200.00 |
V4-3 | 10.30 | 15.50 | V4-3 | 0.00 | 2.50 | 2.00 | 20.00 | 159.65 |
V4-4 | 6.18 | 15.50 | V4-4 | 0.00 | 2.50 | 2.00 | 20.00 | 95.79 |
V4-5 | 11.60 | 15.50 | V4-5 | 0.00 | 2.50 | 2.00 | 20.00 | 179.80 |
V4-6 | 56.20 | 7.00 | V4-6 | 8.50 | 2.50 | 2.00 | 20.00 | 393.40 |
V4-7 | 14.00 | 14.29 | V4-7 | 1.21 | 2.50 | 2.00 | 20.00 | 200.00 |
V4-8 | 25.00 | 8.00 | V4-8 | 7.50 | 2.50 | 2.00 | 20.00 | 200.00 |
V4 reaction mix
Components | Volume (ul) | 8.5x |
10X AmpLigase Buffer | 2.00 | 17.00 |
GP1_V1 (4.54ng/ul) | 2.50 | 21.25 |
Toal | 4.50 |
GP1_V6 set up
Bis-cvt samples | Conc. (ng/ul) | Volume for 200ng (ul) | Bis-cvt samples | H2O (ul) | GP1_V6 | 10X AmpLigase Buffer | Total (ul) | Converted DNA amount |
V6-1 | 17.00 | 11.76 | V6-1 | 5.40 | 0.84 | 2.00 | 20.00 | 200.00 |
V6-2 | 12.30 | 16.00 | V6-2 | 1.16 | 0.84 | 2.00 | 20.00 | 196.80 |
V6-3 | 12.30 | 16.00 | V6-3 | 1.16 | 0.84 | 2.00 | 20.00 | 196.80 |
V6-4 | 14.10 | 14.18 | V6-4 | 2.98 | 0.84 | 2.00 | 20.00 | 200.00 |
V6-5 | 15.40 | 12.99 | V6-5 | 4.17 | 0.84 | 2.00 | 20.00 | 200.00 |
V6-6 | 22.50 | 8.89 | V6-6 | 8.27 | 0.84 | 2.00 | 20.00 | 200.00 |
V6-7 | 7.12 | 16.00 | V6-7 | 1.16 | 0.84 | 2.00 | 20.00 | 113.92 |
V6-8 | 20.00 | 10.00 | V6-8 | 7.16 | 0.84 | 2.00 | 20.00 | 200.00 |
V6 reaction mix
Components | Volume (ul) | 8.5x |
10X AmpLigase Buffer | 2.00 | 17.00 |
GP1_V1 (7.96ng/ul) | 0.84 | 7.14 |
Toal | 2.84 |
Program -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h -> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 6h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN Mix
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume (ul), total 35ul |
AmpliTaq Stoffel | 10 | U/ul | 2 | U/ul | 7.00 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 3.50 |
dNTP | 1 | mM | 100 | uM | 3.50 |
10x AmpLigase Buffer | 10 | x | 1 | x | 3.50 |
H2O | 17.50 |
Amplification
PCR TEST
Components | 1x rxn | 17 x rxn mix |
Captured template | 2.50 | 0.00 |
10uM AmpF6.4Sol | 0.50 | 8.50 |
10uM AmpR6.3 | 0.50 | 8.50 |
2X KAPA SYBR MM | 12.50 | 212.50 |
H2O | 9.00 | 153.00 |
Total | 25.00 |
- - Aliquot 22.5, add 2.5ul of template
PCR with barcoded primers
- - Ind1-16, 15% template
Components | 1x rxn | 17 x rxn mix |
Captured template | 15.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 34.00 |
10uM AmpR6.3.IndX (X=1-16) | 2.00 | 0.00 |
2X KAPA SYBR MM | 50.00 | 850.00 |
H2O | 31.00 | 527.00 |
Total | 100.00 |
- - Aliquot 83, add 2ul of AmpR6.3.IndX, 15ul of template
PAGE quantification
- - Loaded XX ul of each sample
GP1V4
File:ZhangLab 2 2013-12-25 19hr 44min GP1 V4 1-8 PQ.jpg
GP1V6
File:ZhangLab 2 2013-12-25 19hr 46min GP1 V6 1-8 PQ.jpg
PAGE size-selection
File:ZhangLab 2 2013-12-26 13hr 29min PAGE-SS V4.jpg File:ZhangLab 2 2013-12-26 13hr 42min PAGE-SS V6.jpg