Noi/NOTES/2011-3-18

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  • Link to calendar: [[1]]

DMR220k capture (Probe set S1-S3 plus Exp1-Exp3) and library construction of cell samples from Belmonte's lab[edit]

  • continued form 2011_03_10: [[2]]

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 Concentration in the Qubit Unit uL used Dilution Sample Concentration Unit Final conc. (ng/ul) Yields (ng) in 15ul
1. HGPS 75.80 ng/mL 1.00 200.00 75.80 ng/mL 15.16 227.40
2. cHGPS1 90.30 ng/mL 1.00 200.00 90.30 ng/mL 18.06 270.90
3. cHGPS40 68.50 ng/mL 1.00 200.00 68.50 ng/mL 13.70 205.50
4. AWS 55.30 ng/mL 1.00 200.00 55.30 ng/mL 11.06 165.90
5. cAWS1 66.10 ng/mL 1.00 200.00 66.10 ng/mL 13.22 198.30


Capture set up[edit]

Probe calculation[edit]

  • From Parkinson's patient sample sequencing, Dr. Zhang suggested me to adjust the ratio of each subset of the probe. In this study and future study, I will use DMR 220k set S1-S3 (keep the same captured efficiency coefficient) and Exp1-3 by using the same ratio as S2 set. Then set S2 or S3 set as the baseline. In this experiment I set S2 as the baseline.
  • Probe :target ration = 100:1
  • gDNA template amount 200ng
Probes Size Amount of probe requited
(ng)
Multiplier D Actual amount of probe required (ng)
amount of probe required x Multiplier D
Conc. (ng/ul) Probe volume (ul) 6.2 rxn mix
DMR220k.S1 6500.00 2.23 0.56 1.25 2.00 0.62 3.87
DMR220k.S2 6500.00 2.23 1.00 2.23 2.00 1.12 6.91
DMR220k.S3 55000.00 18.90 2.78 52.54 23.20 2.26 14.04
DMR220k.Exp1 55000.00 18.90 1.00 18.90 33.00 0.57 3.55
DMR220k.Exp2 55000.00 18.90 1.00 18.90 25.00 0.76 4.69
DMR220k.Exp3 55000.00 18.90 1.00 18.90 29.00 0.65 4.04
Total volume 5.98 37.10


Sample ID Sample code Conc. (ng/ul) Volume (ul) H2O Total amount of bis-cvt gDNA probe mix (ul) 10x AmpLigase buffer (ul) 105nM Oligo suppressor (ul) H20 Total volume
1. HGPS BM1 15.16 13.50 0.00 204.66 5.98 2.30 1.00 0.22 23.00
2. cHGPS1 BM2 18.06 11.00 2.50 198.66 5.98 2.30 1.00 0.22 23.00
3. cHGPS40 BM3 13.70 13.50 0.00 184.95 5.98 2.30 1.00 0.22 23.00
4. AWS BM4 11.06 13.50 0.00 149.31 5.98 2.30 1.00 0.22 23.00
5. cAWS1 BM5 13.22 13.50 0.00 178.47 5.98 2.30 1.00 0.22 23.00
6. NTC NTC NA 0.00 13.50 0.00 5.98 2.30 1.00 0.22 23.00


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_03_19)      
   -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN preparation[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (17ul)
AmpliTaq Stoffel 10.00 U/ul 2.00 U/ul 3.40
AmpLigase 5.00 U/ul 0.50 U/ul 1.70
dNTP 1.00 mM 100.00 uM 1.70
10x AmpLigase Buffer 10.00 x 1.00 x 1.70
H2O 8.50
aliquot 2.5ul to each tube


PCR Amplification[edit]

(2011_03_21)

  • amplify 2tubes/ sample
  • 5samples x 2 = 10 plus 1 NTC = 11rxn
Reagents volume (ul) 11.5 rxn mix
captured template 9.00 0.00
100uM AmpFV6.3NH2 0.20 2.30
100uM AmpRV6.3NH2 0.20 2.30
50x SYBG 0.40 4.60
2x PhusionHF MM 50.00 575.00
H2O 40.20 462.30
Total volume (ul) 100.00 aliquot 91ul, add 9ul of captured DNA


Program

   98C 30sec 
   (98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles
   72C 2min  
   15C hold.

File:2011 03 21 AmpFR63NH2 rawdata.PNG
Ct value

  • HGPS : 13.89
  • cHGPS1: 12.87
  • cHGPS40: 13.02
  • AWS: 12.84
  • cAWS1: 16.59
  • purify with 0.7vol. Ampure beads, elute with 25ul EB/tube, so total volume = 50ul
  • PAGE quantify DNA concentration (loaded sample 1ul)

File:ZhangLab 2 2011-03-21 21hr 54min AmpFR63NH2 1ul.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)
1. HGPS 8.71 48.00 417.96 6.00 0.19 6.00 60.19
2. cHGPS1 8.84 48.00 424.30 6.00 0.19 6.00 60.19
3. cHGPS40 9.20 48.00 441.75 6.00 0.19 6.00 60.19
4. AWS 11.60 48.00 556.63 6.00 0.19 6.00 60.19
5. cAWS1 9.95 48.00 477.61 6.00 0.19 6.00 60.19


Reagents 1x 5.5 rxn mix
10X NEB buffer4 6.00 33.00
32mM SAM 0.19 1.05
MmeI 6.00 33.00
Total 12.19 aliquot 12.19ul,
add 48ul of amplified amplicon


  • incubate 37C 1hr
  • purify with 0.7vol. Ampure beads, elute with 30ul EB
  • PAGE quantify DNA concentration (loaded sample 2ul)

File:ZhangLab 2 2011-03-22 00hr 48min MmeI digestion.jpg

Sample ID Conc of MmeI completely digested DNA (ng/ul) Total volume (ul) Total amount (ng)
1. HGPS 4.27 30.00 128.05
2. cHGPS1 5.46 30.00 163.91
3. cHGPS40 4.95 30.00 148.48
4. AWS 5.55 30.00 166.59
5. cAWS1 5.28 30.00 158.29


N2 adaptor ligation set up[edit]

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
Sample ID conc.(ng/ul) Volume (ul) for 100ng 20uM N2 adaptors volume (ul) 5xQuickLigase buffer QuickLigase (ul) H2O (ul) Total (ul)
1. HGPS 4.27 23.43 0.40 8.00 2.00 6.17 40.00
2. cHGPS1 5.46 18.30 0.40 8.00 2.00 11.30 40.00
3. cHGPS40 4.95 20.21 0.40 8.00 2.00 9.39 40.00
4. AWS 5.55 18.01 0.40 8.00 2.00 11.59 40.00
5. cAWS1 5.28 18.95 0.40 8.00 2.00 10.65 40.00
6. NTC NA 0.00 0.40 8.00 2.00 29.60 40.00


Reagents 1x 6.5 rxn mix
5xQuickLigase buffer 8.00 52.00
QuickLigase 2.00 13.00
N2 Adapters, conc. (Gr. A, B or C) 0.40 2.60
Total 10.40 aliquot 10.4ul,
add MmeI digested DNA,
adjust volume with H2O


  • incubate at RT for 15 min
  • purify with 1 vol. Ampure beads, elute with 30ul EB

Amplification[edit]

  • Use 1% N2 ligated DNA template for amplification
  • 2tubes/sample: 5x2 = 10rxn plus 1NTC = 11rxn
Reagents 1 rxn 11.5rxn mix
N2 adapter ligated template 1.00 0.00
10uM PCR_F 1.00 11.50
10uM PCR_R.N2 IndX(X= 8,9,10,…,12) 1.00 0.00
50x SYBG 0.40 4.60
2x PhusionHF MM 50.00 575.00
H2O 46.60 535.90
Total volume (ul) 100.00 aliquot 98ul,
add 1ul template,
1ul PCR_R.N2 IndX


Sample IDs IndX
1. HGPS PCR_R.N2Ind8
2. cHGPS1 PCR_R.N2Ind9
3. cHGPS40 PCR_R.N2Ind10
4. AWS PCR_R.N2Ind11
5. cAWS1 PCR_R.N2Ind12
6. NTC PCR_R.N2Ind12


Program

   98C 30sec 
   (98C 10sec -> 64C 20sec-> 72C 30sec) x 11 cycles
   72C 2min  
   15C hold.

File:2011 03 21 N2Amp rawdata.PNG

  • Purify amplified amplicon with 0.7volume Ampure beads, elute with 50ul EB total
  • PAGE quantify DNA concentration (loaded sample 1ul)

File:ZhangLab 2 2011-03-22 05hr 04min PQ SeqLib (1).jpg

Sample IDs IndX Conc. (ng/ul) Total volume (ul) Total amount (ng) Volume for 65ng (ul)
1. HGPS PCR_R.N2Ind8 1.59 50.00 79.36 40.95
2. cHGPS1 PCR_R.N2Ind9 1.89 50.00 94.67 34.33
3. cHGPS40 PCR_R.N2Ind10 1.74 50.00 87.08 37.32
4. AWS PCR_R.N2Ind11 2.00 50.00 100.06 32.48
5. cAWS1 PCR_R.N2Ind12 1.35 50.00 67.33 48.27


  • perform PAGE size selection with 6% TBE 5-well gel (1gels)

File:ZhangLab 2 2011-03-22 06hr 24min PAGE-SS.jpg

  • Total volume of of sequencing libraries: 30ul

File:ZhangLab 2 2011-03-22 05hr 04min PAGE-SS SeqLib-HL091.jpg

  • 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 4.62ng/ul, 22.6nM (336bp), yields = 4.62ng/ul * 30ul = 138ng
  • Sequencing: Sample ID: NP-220K-Belmonte_Ind8-12-March10, HL091 run
  • In the same gel, ALice's samples were included (loaded sample 1ul)

File:ZhangLab 2 2011-03-22 16hr 13min PQ SeqLib PAGE-SS AL.jpg

  • AL1: conc. = 61.03ng/ul
  • AL4: conc. = 51.39ng/ul
  • AL6: conc. = 68.47ng/ul
  • AL8: conc. = 12.52ng/ul
  • Note: Since sample cAWS1 had the captured PCR curve with high Ct value (similar to some sample from PD samples which had low quality), so Dr. Zhang concerns about the quality of this sample. However he suggested me to move on sequencing, and in the next batch of sample include cAWS1 and use 4x more bisulfite converted DNA (but do the bisulfite conversion with 1ug of gDNA per column).