AlanFung:LabNotes/Capturing/2012-5-15

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Sample List[edit]

ChIPK36me3 GM12878
ChIPK36me3 P10
ChIPK36me3 DF2
ChIPK36me3 H0
ChIPK36me3 H7
ChIPK36me3 H14
ChIPK36me3 H16
ChIPK27me3 GM12878
ChIPK27me3 P10
ChIPK27me3 DF2
ChIPK27me3 H0
ChIPK27me3 H7
ChIPK27me3 H14
ChIPK27me3 H16
WCE GM12878
WCE P10
WCE DF2
WCE H0
WCE H7
WCE H14
WCE H16
GM cDNA
P10E3 cDNA
DF2 cDNA
H0 cDNA
H7 cDNA
H14 cDNA
H16 cDNA
GM gDNA
P10E3 gDNA
DF2 gDNA
H0 gDNA
H7 gDNA
H14 gDNA
H16 gDNA
GmgDNA:H0gDNA::1:1 38

Sample[edit]

  • H7 and H14 gDNA has the most amount and I would like to use these two samples for a first test
Sample ID User ID Date Time ng/ul A260 A280 260/280 260/230 Constant Cursor Pos. Cursor abs. 340 raw Vol Total to get 1000ng Leftover for me
H7 gDNA Default 4/5/2012 3:57 PM 184.33 3.687 1.787 2.06 2.23 50 230 1.652 0.001 100 18433 5.43 94.6
H14 gDNA Default 4/5/2012 3:58 PM 167.18 3.344 1.669 2 2.17 50 230 1.541 0.016 100 16718 5.98 94.0

Probes Needed[edit]

  • Amount of probes
  • From the gel quantification we have 29.47ng/ul x 80ul =2.36ug of probes
  • Probe:target ratio: 200:1
H7 184.33ng/ul * 1.09ul = 200ng
H14 167.18ng/ul * 1.20ul = 200ng


To capture the 200ng DNA

  • Total probe molecule
  • 200ng/3pgx200x100,000
  • Mol of probe= 200ng/3pg*200*100,000/(6*10^23)
  • Concentration of probe set is 29.47 ng/ul.

The length of the probe is approximately 100 base.

Calculate probe concentration in nM[edit]

  • Since 1base = 330Da (for single stranded DNA, 660Da for ds),
  • the concentration of probe is *29.47ng/ul*1x10^6ul/L*bp mol/330g*1/100bp = 893nM
  • the volume the probe needed is 200ng/3pg x 200 x 100,000 / {(6x10^23)x 893 nM} = 2.49 ul

Capture System Setup[edit]

Content Volume Content Volume
H7 gDNA 1.09 H14 gDNA 1.20
10X Ampligase Buffer 4 10X Ampligase Buffer 4
CES36K 2.49 CES36K 2.49
H2O 12.42 H2O 12.31
Total 20 Total 20
95C 2min -> -0.02C/sec to 60C -> 60C 24h 
   -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
   -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul Exo I&III
   -> 37C 2h -> 90C 5min -> 4C hold