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