Dinh/NOTES/2010-8-22: Difference between revisions

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| AmpF6.3NH2||/5AmMC6/CAGATGTTATCGAGGTCCGAC||5' Amino modifier C6
| AmpF6.3NH2||/5AmMC6/CAGATGTTATCGAGGTCCGAC||5' Amino modifier C6
|-
|-
| AmpR6.3NH2||/5AmMC6/GGAACGATGAGCCTCCAC||5' Amino modifier C6
| AmpR6.3NH2||/5AmMC6/GGAACGATGAGCCTCCAAC||5' Amino modifier C6
|-
|-
| PE_t_N2, Y-adapter for MmeI ligation-top ||ACACTCTTTCCCTACACGACGCTCTTCCGATCTN*N||3'-Phosphorothioate bond
| PE_t_N2, Y-adapter for MmeI ligation-top ||ACACTCTTTCCCTACACGACGCTCTTCCGATCTN*N||3'-Phosphorothioate bond

Latest revision as of 00:48, 12 November 2010

BSPP Capture and N2 library construction of John Hopkins Tumor Samples[edit]

Materials[edit]

Reagents and Kits Source Cat#
EZ DNA Methylation Gold Kit Zymo Research D5005
10x Ampligase buffer Epicentre A1905B
Ampligase Epicentre A3210K
dNTP New England Biolab N0447L
Stoffel fragment Applied Biosystems N808-0038
ExoI Epicentre X40520K
ExoIII Epicentre EX4425K
2xPhusion HF Master Mix New England Biolab F-531L
MmeI New England Biolab R0637L
Quick Ligation Kit New England Biolab M2200S
SYBR GreenI nucleic acid gel stain Invitrogen S7585
Agencourt AMPure XP Beckman Coulter A63880
100% Ethanol
QIAQuick PCR Purification Kit Qiagen 28106
E-Gel Size Select 2% Invitrogen G6610-02


Materials Source Cat#
Low Tube Strips, CLR BioRad TLS0801
Microseal 'B' Film BioRad MSB1001
Barrier Tips, or low retention pipette tips Neptune BT200, BT10E, BT1000, BT20
Mineral Oil Sigma M5904-500ml
6% TBE Gel 1.0mmx10well Invitrogen EC6265BOX
10x TBE buffer for PAGE National Diagnostics EC-860
SYBR Gold nucleic acid gel stain Invitrogen S11494


Primers Sequence Modifications
AmpF6.3NH2 /5AmMC6/CAGATGTTATCGAGGTCCGAC 5' Amino modifier C6
AmpR6.3NH2 /5AmMC6/GGAACGATGAGCCTCCAAC 5' Amino modifier C6
PE_t_N2, Y-adapter for MmeI ligation-top ACACTCTTTCCCTACACGACGCTCTTCCGATCTN*N 3'-Phosphorothioate bond
PE_b_A, Y-adapter for MmeI ligation-bottom /5Phos/AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-Phosphorylation
PCR_Fs AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC None
PCR_R.N2Ind1 CAAGCAGAAGACGGCATACGAGATCGTGATCTCGGCATTCCTGCTGAACCGCTCTT None
PCR_R.N2Ind2 CAAGCAGAAGACGGCATACGAGATACATCGCTCGGCATTCCTGCTGAACCGCTCTT None
PCR_R.N2Ind3 CAAGCAGAAGACGGCATACGAGATGCCTAACTCGGCATTCCTGCTGAACCGCTCTT None
PCR_R.N2Ind4 CAAGCAGAAGACGGCATACGAGATTGGTCACTCGGCATTCCTGCTGAACCGCTCTT None

Bisulfite Conversion[edit]

Perform bisulfite conversion according to instructions given in EZ Methylation Gold Kit.
Sample ID Volume Used(ul) Conc(ng/ul) Volume obtained(ul) Conc(ng/ul) Yield
499T 4 250 8.5 91.5 0.78
499N 5 195 8.5 67.9 0.59
536T 4 250 8.5 76.2 0.65
536N 4 250 8.5 105.4 0.9
614T 4 250 8.5 94.8 0.81
614N 4 250 8.5 81.4 0.69
Jurkat gDNA 10 249 8.5 212.5 0.73

Capture[edit]

Set up capture reaction in low profile strip tubes (or PCR tubes/plates). 
Spin down the reaction mix and add 1-2 droplets of mineral oil with P200.
Use Microseal film to cover the tubes.

Probe/Target Ratio 100
Probes size 220000
Template 300 ng
Human gDNA MW 1.82E+012 g/mole (3E9bpx607.4D/bp+157.9D)
Human gDNA 1.65E-019 moles
Probes 1.65E-017 moles
Probes MW 6.88E+9 g/mole (220000x103bpx303.7D/bp+79D)
Amount probes required 113 ng
Concentration probes 1 27 ng/ul
Vol 4.20 ul
Concentration probes 2 23 ng/ul
Vol 4.93 ul

Reaction Mix

Sample ID Conc. (ng/ul) Vol(ul) Probes(ul) 10xAmpligase Buffer (ul) H2O Total
Sarven, 499T (biscvt) 91.5 3.28 4.2 1 1.53 10
Sarven, 499N (biscvt) 67.9 4.42 4.2 1 0.39 10
Sarven, Jurkat (biscvt) 212.5 1.41 4.2 1 3.39 10
Dinh, 536T (biscvt) 76.2 3.94 4.9 1 0.16 10
Dinh, 536N (biscvt) 105.4 2.85 4.9 1 1.25 10
Dinh, Jurkat (biscvt) 212.5 1.41 4.9 1 2.69 10

Program

   95c 30sec -> cool down to 58C at 0.02C/sec -> 58C 20h 
   -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) 
   -> 58C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 1h -> 94C 2min -> 4C hold.
Adding 2ul SLN mix after >20 h of 58C incubation:
Leave tubes in thermocycler and just remove Microseal film cover to add SLN mix.
Use P10, low retention tips and make sure to reach the bottom of the tube before dispensing.
Dispense slowly, then slowly pull out the pipette tip before releasing hold on dispenser.
Ensure that all reagents were pumped out. 
Cut new Microseal films to cover the tubes.
Adding 2ul ExoI/III mix after 3-4 h of 58C incubation:
Perform same steps as adding SLN. 
SLN Mix 1x 16x Final Conc
H2O 0.55 8.8
dNTP(1mM) 0.05 0.8 50uM
10xAmpligase Buffer 0.1 1.6 1x
AmpLigase 0.1 1.6 0.5U/ul
Stoffel 0.2 3.2 2U/ul
TOTAL 1 16

For ExoI/III mix, add equal volumes of Exo I and Exo III.

PCR Amplification[edit]

3 + 1 NTC = 4

Reagent Vol (1x) Vol (8x)
Template (*) 10
2x PhusionHF MM 50 400
AmpF6.3NH2(10uM) 2 16
AmpR6.3NH2(10uM) 2 16
50x SYBR Green 0.4 3.2
Rnase Free H2O 35.6 286
TOTAL 100
File:Capture rt-pcr JH- tumor-normal 499 and 536.PNG
File:ZhangLab 2 2010-08-24 JH-tumor samples capture amp.png
Expected capture amplicon size: 300bp

Purify with 0.9x AMPure beads, elute with 40ul EB

Mme I digestion[edit]

1 units per 1ug phiX 174 DNA
1 phiX = 5 sites/5389bp
1 amplicon = 2 sites/300bp

7.2 units MmeI to digest 1ug capure PCR amplicons => 2.16 units to digest 300ng.
  VOL     REAGENT      Conc            Final
30.6 ul DNA + H2O                      ~300ng
 4.0 ul NEBuffer 4      10x             1x 
 2.4 ul MmeI         2 units/ul        4.8 units (add double amount of required MmeI)
 3.0 ul SAM             1mM            75uM
 -------------------------------------------- 
40.0 ul TOTAL
Incubate at 37C for 1 hr. Purify with 1 Qiaquick column each, elute with 30ul EB.
Run 1 ul each on 6% TBE gel
File:ZhangLab 2 2010-08-25 JH-tumor 499 536 MmeI digest.png
Expected digest size: 300-43-45=212bp.
Quantification with low mass ladder
' Signal Intensity Volume Loaded Calculated amount DNA(ng)
0.1x low mass ladder 8966.52 3 0.75
0.1x low mass ladder 15987.17 6 1.5
5T partially digested 5545.29 1 0.38
5T fully digested 2309.56 1 bad
5N partially digested 2578.99 1 0.07
5N fully digested 974.66 1 bad
4T partially digested 5612.13 1 0.39
4T fully digested 4521.19 1 0.28
4N partially digested 912.56 1 bad
536T -> 80% of partially digested band, 0.38ng/1ul*0.8*20 = ~6ng
536N -> 80% of partially digested band, 0.07ng/1ul*0.8*20 = ~1ng
499T -> 0.28*20 = ~6ng
499N -> est. = to 536N based on gel = ~1ng

N2 Adapter Ligation[edit]

Adaptors preparation:

 20ul PE_N2_adaptor (100uM)
 20ul PE_b_A (100uM)
 10ul Stoffel buffer (10x)
 50ul H2O
 ----------------------------
 100 ul TOTAL (20uM adaptors)

Program

94C 2min -> 0.2C/sec to 20C -> 4C hold

Adapters to ligated product ratio: 20:1

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 * 20:1 = 0.02 nmole adapters required.
Adapters (ul) = 0.02nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.02nmoles/ (20E-3 nmoles/ul) = 0.77 ul 
-----> 0.77 ul of 20uM adapters per 100ng digested products. 
-----> 15.4 ul of 1uM adapters per 100ng digested products.
use 1 ul of 1uM adapters per 6 ng digested products.
use 0.4 ul of 0.5uM adapters per 1 ng digested products. 
 SampleID   DNA(ul)          Adapters(ul)          QuickLigase(ul)     2xQuickLigase Buffer(ul)
 499T       20ul (~6ng)        1 (1uM)               1                   22     
 499N       20ul (~1ng)      0.4 (0.5uM)             1                   21.4
 536T       20ul (~6ng)        1 (1uM)               1                   22
 536N       20ul (~1ng)      0.4 (0.5uM)             1                   21.4
 
Incubate at RT fo 15 min. Purify with 0.7x AMPure beads. Elute with 40ul EB.

Amplification[edit]

  1x    Reagents
10.0ul  adapter ligased DNA
 2.0ul  PCR_F(10uM) 
 2.0ul  PCR_R.N2IndX(10uM) [X=1,2,3,4]
 0.4ul  SYBR Green 50x
35.6ul  H2O
50.0ul  Phusion HF, 2xMM
--------------------------------
100 ul each x 3 per sample

Program

98C - 30s, (98C - 10s, 62C - 20s, 70C - 30s)x10/12, 72C - 2min.

File:Ligated product rt-pcr JH- tumor-normal 499 and 536.PNG
File:Ligated product rt-pcr JH- tumor-normal 499 and 536 -2.PNG

Purify with 2 Qiaquick columns for each sample (~145ul reaction mix + 600ul PB per column)
Yield: 536T (56.5ng/ul), 499T (61.9ng/ul), 536N (12.7ng/ul), 499N (13.3ng/ul)
File:ZhangLab 2 2010-08-25 JH-tumor 499 536 Ligated products.png
Expected ligated product size: 212+36+36=284bp.
Expected amplified ligated product size: 212+64+60=336bp.

Size selection[edit]

Pool 254 ng of each sample into one tube --> 20ul (536N), 19.1ul (499N), 4.10ul(499T), 4.50ul(536T).
Load sample into 2 lanes, use 25bp ladder ( should use low mass ladder instead to see 200-400bp range clearer ).
Run E-Gel on DC mode (13 min). 
Remove sample when it reaches bottom well once.
File:ZhangLab 2 2010-08-25 JH-tumor 499 536 ss.png
Name on Tube:                       Library ID:
DD-BSPP-JH.499,536-Aug25                  DD-BSPP220K1-JH,499T.499N.536T.536N-Aug25-N2barcoded

Discussion[edit]

  • Total RT-PCR cycles: 25-30 for entire protocol.
  • Notes on MmeI use says excess MmeI blocks cleavage and advises stoichiometric concentrations be used for optimal cleavage.
  • ~1ng MmeI digestion product is enough for ligation.