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