Noi/NOTES/2013-12-24: Difference between revisions

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** V4 set: conc. ssDNA (ND) = 34.6ng/ul, yield = 37.5ul * 34.6ng/ul = 1.3ug
** 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
** V6 set: conc. ssDNA (ND) = 49.7ng/ul, yield = 37.5ul * 49.7ng/ul = 1.86ug
* I will finish up probe prep later
== Removal of amplification adapters ==
* Set up the same volume for both V4 & V6
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1x rxn'''
|-
| ssDNA ||37.50
|-
| USER||2.50
|-
| Total||40.00
|}
* 1.30ug of V4 in total 40ul & 1.86ug of V6 in total 40ul
* Incubate at 37C for 3hr
* Add 2.5ul of RE-DpnII-V4 guide oligo to GP1_V4 and 2.5ul of RE-DpnII-V6 guide oligo to GP1_V6. Mix well and spin down.
* Incubated at 94 C for 2 min, cool down temperature to 37C for 3 min
* Add 2.5ul of DpnII to each tube. Mix well by pipetting up and down 10x while the tube on thermocycler.
* Incubate at 37C for 2hr
* Heat inactivate at 75C for 20min
* Purify with 1x QIAquick column for each set
** I add 700ul of PB buffer, just add extra volume of buffer to fill up the column)
* Elute with 60ul EB buffer for each set
* Measure concentration with ND (ssDNA, After elution and ND measurement there is ~58ul left total)
** GP1V4 set: conc. ssDNA (ND) = 13.1ng/ul, total yield = 58ul * 13.1ng/ul = 760ng
** GP1V6 set: conc. ssDNA (ND) = 18.9ng/ul, total yield = 58ul * 18.9ng/ul = 1,096ng
GP1_V4 GP1_V6
* Perform PAGE size-selection (1x 2D well for each set)
[[File:ZhangLab_2 2013-12-22 17hr 34min_GP1_V4V6_PAGE-SS.jpg|600px]]
** Resuspend with 900ul 1X TE buffer in 2mL LoBind tube
** After incubation for 1hr, pass through 2x nanosep and split ~400 to 2x tube for each set.
** Precipitate with 1.34ul glycoblue, 40ul 3M NaOAc pH 5.5, and 1mL 100% EtOH
** Incubate at -80C O/N
** Spin down and resuspend 11ul each tube (total 22ul for each set)
* Measure concentration with Qubit ssDNA quantification (1ul for the assay)
** GP1V4 set: conc. ssDNA (ND) = 4.54ng/ul, total yield = 20ul * 4.54ng/ul = 90.8ng
** GP1V6 set: conc. ssDNA (ND) = 7.96ng/ul, total yield = 20ul * 7.96ng/ul = 159.2ng
* Verify in 6% TBU gel (load 0.5ul)
[[File:ZhangLab_2 2013-12-23 16hr 30min_PG1-gelVerification.jpg| 400px]]
 
----
 
= BSPP capture =
= BSPP capture =
* Alan extracted gDNA of 5 cancer cell lines. He split the cell into duplicates, so there were two tubes of purified gDNA.
* Alan extracted gDNA of 5 cancer cell lines. He split the cell into duplicates, so there were two tubes of purified gDNA.

Latest revision as of 19:29, 17 May 2014

BSPP capture of GP1 samples[edit]

  • 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[edit]

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

Removal of amplification adapters[edit]

  • Set up the same volume for both V4 & V6
Components 1x rxn
ssDNA 37.50
USER 2.50
Total 40.00
  • 1.30ug of V4 in total 40ul & 1.86ug of V6 in total 40ul
  • Incubate at 37C for 3hr
  • Add 2.5ul of RE-DpnII-V4 guide oligo to GP1_V4 and 2.5ul of RE-DpnII-V6 guide oligo to GP1_V6. Mix well and spin down.
  • Incubated at 94 C for 2 min, cool down temperature to 37C for 3 min
  • Add 2.5ul of DpnII to each tube. Mix well by pipetting up and down 10x while the tube on thermocycler.
  • Incubate at 37C for 2hr
  • Heat inactivate at 75C for 20min
  • Purify with 1x QIAquick column for each set
    • I add 700ul of PB buffer, just add extra volume of buffer to fill up the column)
  • Elute with 60ul EB buffer for each set
  • Measure concentration with ND (ssDNA, After elution and ND measurement there is ~58ul left total)
    • GP1V4 set: conc. ssDNA (ND) = 13.1ng/ul, total yield = 58ul * 13.1ng/ul = 760ng
    • GP1V6 set: conc. ssDNA (ND) = 18.9ng/ul, total yield = 58ul * 18.9ng/ul = 1,096ng

GP1_V4 GP1_V6

  • Perform PAGE size-selection (1x 2D well for each set)

File:ZhangLab 2 2013-12-22 17hr 34min GP1 V4V6 PAGE-SS.jpg

    • Resuspend with 900ul 1X TE buffer in 2mL LoBind tube
    • After incubation for 1hr, pass through 2x nanosep and split ~400 to 2x tube for each set.
    • Precipitate with 1.34ul glycoblue, 40ul 3M NaOAc pH 5.5, and 1mL 100% EtOH
    • Incubate at -80C O/N
    • Spin down and resuspend 11ul each tube (total 22ul for each set)
  • Measure concentration with Qubit ssDNA quantification (1ul for the assay)
    • GP1V4 set: conc. ssDNA (ND) = 4.54ng/ul, total yield = 20ul * 4.54ng/ul = 90.8ng
    • GP1V6 set: conc. ssDNA (ND) = 7.96ng/ul, total yield = 20ul * 7.96ng/ul = 159.2ng
  • Verify in 6% TBU gel (load 0.5ul)

File:ZhangLab 2 2013-12-23 16hr 30min PG1-gelVerification.jpg


BSPP capture[edit]

  • 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[edit]

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[edit]

Sample IDs and labeling matching[edit]

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[edit]

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[edit]

Probe calculation[edit]

GP1_V4[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

PCR TEST[edit]

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[edit]

- 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
Samples Index
BXPC3_V4 Ind_1
U87MG_V4 Ind_2
PANC1_V4 Ind_3
BE(2)C_V4 Ind_4
T98G_V4 Ind_5
UCLA-SZ_B1_V4 Ind_6
UCLA-SZ_D1_V4 Ind_7
UCLA-SZ_H11_V4 Ind_8
BXPC3_V6 Ind_9
U87MG_V6 Ind_10
PANC1_V6 Ind_11
BE(2)C_V6 Ind_12
T98G_V6 Ind_13
UCLA-SZ_B1_V6 Ind_14
UCLA-SZ_D1_V6 Ind_15
UCLA-SZ_H11_V6 Ind_16

PAGE quantification[edit]

- Loaded 5ul of each sample (total 60ul after bead purification) in 6% TBE gel

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

Sample ID Conc. (ng/ul) Yields in 55ul (ng) Volume for 30ng
GP1-V4-1 2.13 116.92 14.11
GP1-V4-2 1.57 86.61 19.05
GP1-V4-3 1.40 77.11 21.40
GP1-V4-4 1.45 79.51 20.75
GP1-V4-5 1.60 88.00 18.75
GP1-V4-6 1.60 88.01 18.75
GP1-V4-7 0.56 30.74 53.67
GP1-V4-8 1.12 61.45 26.85
Sample ID Conc. (ng/ul) Volume (ul)
GP1-V4-1 0.190907387 20
GP1-V4-2 N/A 50
GP1-V4-3 N/A 50
GP1-V4-4 N/A 50
GP1-V4-5 N/A 50
GP1-V4-6 0.001140051 40
GP1-V4-7 N/A 50
GP1-V4-8 0.08565092 40

PAGE size-selection[edit]

GP1V4
File:ZhangLab 2 2013-12-26 13hr 29min PAGE-SS V4.jpg
GP1V6
File:ZhangLab 2 2013-12-26 13hr 42min PAGE-SS V6.jpg

Sequencing library IDs[edit]

GP1V4: NP-GP1V4-Ind1_16-Dec26, 2013
GP1V6: NP-GP1V6-Ind1_16-Dec26, 2013
Sequence with MiSeq run: M01186, PE 151+6+151bp