Jie:LabNotes/CpgSeq/2009-7-1

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parkinson's patient DNA sample capture by cpg97k[edit]

Bisulfite conversion of Parkinson's patient DNA[edit]

No sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
Par_1 5028 320ng/ul x 1 tube 7ul 13ul 130ul 42.8ng/ul x 10ul
Par_2 4735 260ng/ulx 1 tubes 8ul 12ul 130ul 46.9ng/ul x 10ul
Par_3 4789 280ng/ulx 1 tubes 7ul 13ul 130ul 31.2ng/ul x 10ul
Par_4 5171 420ng/ulx 1 tubes 5ul 15ul 130ul 41.8ng/ul x 10ul
Par_5 1818 480ng/ulx 1 tubes 4.5ul 15.5ul 130ul 21.1ng/ul x 10ul
Par_6 1947 360ng/ul x 1 tubes 6ul 14ul 130ul 55ng/ul x 10ul
Par_7 1741 560ng/ul x 1 tube 4ul 16ul 130ul 27.3ng/ul x 10ul
Par_8 4977 260ng/ulx 1 tube 8ul 12ul 130ul 38.4ng/ul x 10ul
Par_9 4879 340ng/ulx 1 tube 6ul 14ul 130ul 62.1ng/ul x 10ul
Par_10 4526 380ng/ulx 1 tube 6ul 14ul 130ul 34.6ng/ul x 10ul
  • Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent.
  • Add 130ul of CT conversion reagent to samples.
  • 98C 10min -> 64C 150min -> 4c overnight.
  • Add 600ul M-Binding buffer to spin columns.
  • Add the converted samples to the columns, close the gap and mix by inverting several times.
  • Spin at 15,000rpm for 30sec.
  • Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
  • Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
  • Add 200ul M-Wash Buffer, spin for 30 sec.
  • Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
  • Measure the DNA with Nanodrop:RNA

I got low concentration after bis-conversion. i quantified the gDNA concentration with nanodrop and redo the bis-conversion.

No sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
Par_1 5028 111.9ng/ul x 1 tube 19ul 1ul 130ul 90.8ng/ul x 10ul
Par_2 4735 150.5ng/ulx 1 tubes 14ul 6ul 130ul 86.7ng/ul x 10ul
Par_3 4789 118.7ng/ulx 1 tubes 19ul 1ul 130ul 74ng/ul x 10ul
Par_4 5171 143.9ng/ulx 1 tubes 14ul 6ul 130ul 98.3ng/ul x 10ul
Par_5 1818 113.5ng/ulx 1 tubes 19ul 1ul 130ul 69ng/ul x 10ul
Par_6 1947 151.4ng/ul x 1 tubes 14ul 6ul 130ul 109.7ng/ul x 10ul
Par_7 1741 133ng/ul x 1 tube 18ul 2ul 130ul 84.5ng/ul x 10ul
Par_8 4977 92.7ng/ulx 1 tube 20ul 0ul 130ul 73.4ng/ul x 10ul
Par_9 4879 190.6ng/ulx 1 tube 11ul 9ul 130ul 110.3ng/ul x 10ul
Par_10 4526 102.5ng/ulx 1 tube 20ul 0ul 130ul 83.1ng/ul x 10ul

capture the samples with two cpg97k subsets[edit]

The probe set was prepared by Alan.

No sample sample concentration 10xLigase buffer template+cpg97k_A(25.6ng/ul_06/18) vol+suppress oligo+H2O template+cpg97k_B(21ng/ul_06/18) vol+suppressor oligo+H2O
Par_1 5028 90.8ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_2 4735 86.7ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_3 4789 74ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_4 5171 98.3ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_5 1818 69ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_6 1947 109.7ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_7 1741 84.5ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_8 4977 73.4ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_9 4879 110.3ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
Par_10 4526 83.1ng/ul 1ul 4+2.5ul+1ul+1.5ul 4+2ul+1ul+2ul
positive control Jurkat bis-gDNA 200ng/ul 1ul 1+1ul+1ul+6ul
negative control 0 1ul 0+1ul+1ul+6ul

dNTP concentration 10mM(NEB)

                                           H2O  Ligase Buffer Ligase  Amplitaq  dNTP           
2U/ul Stoffel; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul    0.1ul    0.1ul   0.2ul    (10mM)0.1ul 
                                       x100  50ul     10ul    10ul     20ul     10ul

PCR[edit]

Template 10ul x11

2X iProof Mastermix     50ul      550ul
AmpF6.3SoL (10uM)        4ul       44ul
AmpR6.3SoL (10uM)        4ul       44ul   
50X SYBG I             0.4ul      4.4ul
H2O                   31.6ul    347.6ul

98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->(98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold.

File:20090703 Parkinson sample cpg97k A.jpg20090703_Parkinson_sample_cpg97k_A

File:20090703 Parkinson sample cpg97k B.jpg20090703_Parkinson_sample_cpg97k_B

I purified all samples with qiaquick column. Nanodrop quantification and mix the subset A and B captured targets with 1:1 ratio:

shotgun library construction[edit]

PCR amplification with AmpF6.3NH2/AmpR6.3NH2 (2009-07-14)[edit]

I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
reaction system                                                 x4     x10  
H2O                                                  45ul      180ul    
2x Mastermix                                         50ul      200ul      
AmpF6.3NH2(10uM)                                      2ul       8ul       
AmpR6.3NH2(10uM)                                      2ul       8ul     
50x SYBG I                                          0.4ul      1.6ul    
template                                      1ul/each for purified cpg97k
Total                                               100ul      800ul     
94C 2min -> (94C 45S -> 55C 45S -> 72C 45S) x 8 ->72C 3 min -> 15C hold.

File:20090714 Parkinson samples with AmpF6.3NH2 reamplification.jpg20090714_Parkinson samples with AmpF6.3NH2 reamplification
I purified the samples with Qiaquick column. Quantify with nanodrop and send the samples to Billy.

Sample Par. ship to Billy[edit]

Sample ID Concentration (ng/ul) Volume (ul) Total Amt. (ng)
Par_1 21.22 30 636.6
Par_2 15.25 30 457.5
Par_3 18.05 30 541.5
Par_4 19.97 30 599.1
Par_5 29.63 30 888.9
Par_6 15.82 30 474.6
Par_7 35.41 30 1062.3
Par_8 46.59 30 1397.7
Par_9 22.29 30 668.7
Par_10 30.4 30 912

endrepair with epicentre kit(2009-07-20)[edit]

100 ul DNA 
 15 ul 10X End-Repair Buffer
 15 ul dNTP Mix
 15 ul ATP
  3 ul End-Repair Enzyme Mix
150 ul Total reaction volume
Incubate at room temperature for 45 minutes. Purify with minelute column.

A tail addition[edit]

                                   x8
  Blunt-ended DNA        10ul      80
  10X Klenow buffer      1.5ul     12
  1mM dATP                3ul      24
  Klenow fragment (exo-)  1ul       8
  37C 30min, purified with MinElute columns, eluted with 20ul EB. 
After A tail, I did size selection for No7 and positive control, then do the adaptor ligation. 
After adaptor ligation, i goes to PCR directly without size selection again.
However, I can only see the positive control product only.
So I skipped the size selection before adaptor ligation for No 1~6, and do the size selection after adaptor ligation.  
However, the expected band after PCR is so weak that I am not sure the targets were successfully amplified.

adaptor ligation[edit]

    DNA                                20ul
    10x QuickLigase buffer (enzymatic)  3ul
    20uM Adaptor oligo mix              3ul
    T4 DNA QuickLigase (enzymatic)      2ul
    Incubate at RT for 15 minutes.
    Purified with Qiaquick columns, eluted with 12ul EB

PCR[edit]

   Template               15ul        
   2x iProof mix          50ul       
   Solexa_PCR_up (10uM)    4ul        
   Solexa_PCR_lo_PE (10uM) 4ul        
   H2O                     30ul       
   50X SYBG I             0.2ul      

File:20090721 shotgun libr par7 positive control.jpg20090721_shotgun_libr_par7_positive control

File:20090721 shotgun libr par1~6.jpg20090721_shotgun_libr_par1~6

redo the shotgun library starting from PCR amplification with AmpF6.3NH2/AmpR6.3NH2 (2009-07-22)[edit]

I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
reaction system                                                 x4       x17  
H2O                                                  50ul      200ul     3400
2x Mastermix                                         50ul      200ul     3400 
AmpF6.3NH2(100uM)                                    0.2ul     0.8ul     13.6 
AmpR6.3NH2(100uM)                                    0.2ul     0.8ul     13.6
50x SYBG I                                          0.4ul      1.6ul     27.2
template                                      1ul/each for purified cpg97k
Total                                               100ul      800ul     
94C 2min -> (94C 45S -> 55C 45S -> 72C 45S) x 8 ->72C 3 min -> 15C hold.
I did the PCR reamplification with Amp6.3 primers with Parkinson's sample 1 and 10 with the same PCR condition.

Sample Par. ship to Covaris (2009-07-27)[edit]

Sample ID Total Amt. (ng)
Par_1 580
Par_2 650
Par_3 580
Par_4 520
Par_5 640
Par_6 500
Par_7 490
Par_8 840
Par_9 590
Par_10 400

shotgun library construction 2009-08-05 refer to LabNotes on [1][edit]