AlanFung:LabNotes/Capturing/2012-8-10: Difference between revisions

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| align="center" | 2
| align="center" | 2
| align="center" | 7.6
| align="center" | 7.6
| align="center" | 50
|- style="font-size:8pt" align="center"
| height="13" | P10
| align="center" | 1.20
| align="center" | 40.00
| align="center" | 0.60
| align="center" | 2
| align="center" | 7.4
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|- style="font-size:8pt" align="center"
| height="13" | DF2
| align="center" | 0.70
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| align="center" | 7.7
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| align="center" | 7.4
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|- style="font-size:8pt" align="center"
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*Make sure we have enough reagent before start,
*need 50ul probes, check
*need 66ul 10X Ampligase Buffer,
*need 33ul dNTP (10uM),
*need 13.2ul dNTP (10uM)
*need 13.2ul Stoffel (10U/ul)
*need 66ul Exo I (20U/ul)
*need 66ul ExoIII (200U/ul)
===Prepare SLN Mix===
*Dilute 1mM dNTP 1 in 100 to 10uM dNTP
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="background-color:#B1A0C7;font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold;text-decoration:underline" width="270" height="13" | SLN Mix for 20ul Rxn
|style="font-weight:bold;text-decoration:none" width="121" | 1x
| width="41" | 16.5x
|- style="font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold" height="13" | dNTP (10uM)
| align="center" | 0.5
| align="center" | 8.25
|- style="font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold" height="13" | Stoffel (10U/ul)
| align="center" | 0.2
| align="center" | 3.30
|- style="font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold" height="13" | Ampligase (5U/ul)
| align="center" | 0.2
| align="center" | 3.30
|- style="font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold" height="13" | H2O
| align="center" | 0.1
| align="center" | 1.65
|- style="font-size:8pt" align="center" valign="bottom"
| align="center" height="13" | &nbsp;
| align="center" | &nbsp;
| align="center" |
|- style="background-color:#B1A0C7;font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold;text-decoration:underline" height="13" | SLN Mix for 50ul Rxn
|style="font-weight:bold;text-decoration:none" | 1x
| 19.8x
|- style="font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold" height="13" | dNTP (10uM)
| align="center" | 1.25
| align="center" | 24.75
|- style="font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold" height="13" | Stoffel (10U/ul)
| align="center" | 0.5
| align="center" | 9.90
|- style="font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold" height="13" | Ampligase (5U/ul)
| align="center" | 0.5
| align="center" | 9.90
|- style="font-size:8pt" align="center" valign="bottom"
|style="font-weight:bold" height="13" | H2O
| align="center" | 0.25
| align="center" | 4.95
|}
===Prepare Exo I and III Mix===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="background-color:#538DD5;font-size:8pt" align="center"
|style="font-weight:bold;text-decoration:underline" width="121" height="13" | Exo I & III for 20ul Rxn
|style="font-weight:bold;text-decoration:none" width="39" | 1X
| width="33" | 16.5x
|- style="font-size:8pt" align="center"
| height="13" | Exo I (20U/ul)
| align="center" | 1
| align="center" | 16.5
|- style="font-size:8pt" align="center"
| height="13" | ExoIII (200U/ul)
| align="center" | 1
| align="center" | 16.5
|- style="font-size:8pt" align="center"
| align="center" height="13" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
|- style="background-color:#538DD5;font-size:8pt" align="center"
|style="font-weight:bold;text-decoration:underline" height="13" | Exo I & III for 50ul Rxn
|style="font-weight:bold;text-decoration:none" | 1X
| 19.8x
|- style="font-size:8pt" align="center"
| height="13" | Exo I (20U/ul)
| align="center" | 2.5
| align="center" | 49.5
|- style="font-size:8pt" align="center"
| height="13" | ExoIII (200U/ul)
| align="center" | 2.5
| align="center" | 49.5


|}
|}

Revision as of 05:32, 12 August 2012

Capturing Remaining Gimelbrant lab Sample

  • Add 2 drops of mineral oil to prevent evaporation
  • Samples stored at tall -20C Alan Rack white box red label "Gimelbrant Lab"
ChIPK36me3 Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 0.90 40.00 0.45 2 7.6 50
H0 1.00 40.00 0.50 2 7.5 50
H7 0.60 40.00 0.30 2 7.7 50
H14 0.50 40.00 0.25 2 7.8 50
H16 0.60 40.00 0.30 2 7.7 50
                
ChIPK27me3 Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 1.20 40.00 0.60 2 7.4 50
H0 2.4 40.00 1.20 2 6.8 50
H7 1.2 40.00 0.60 2 7.4 50
H14 0.5 40.00 0.25 2 7.8 50
H16 1.3 40.00 0.65 2 7.4 50
                
WCE Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM12878 35.39 5.65 2.49 2 9.9 20
P10 35.39 5.65 2.49 2 9.9 20
DF2 35.39 5.65 2.49 2 9.9 20
H0 35.39 5.65 2.49 2 9.9 20
H7 35.39 5.65 2.49 2 9.9 20
H14 35.39 5.65 2.49 2 9.9 20
H16 35.39 5.65 2.49 2 9.9 20
                
cDNA Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
GM 38.12 5.25 2.49 2 10.3 20
H0 38.12 5.25 2.49 2 10.3 20
H7 38.12 5.25 2.49 2 10.3 20
H14 38.12 5.25 2.49 2 10.3 20
H16 38.12 5.25 2.49 2 10.3 20
                
gDNA Concentration ng/ul Vol    Vol of probes 10X Ampligase Buffer H2O Total
H7 38.12 5.25 2.49 2 10.3 20
H14 38.12 5.25 2.49 2 10.3 20
H16 38.12 5.25 2.49 2 10.3 20
  • When adding regents to the tube make sure you penetrate the oil layer
Store under βCES36K
95C 2min -> -0.2C/sec to 60C -> 60C 24h 
   -> add 1ul/2.5ul(for 50ul rxn) SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
   -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul(5ul for 50ul rxn) Exo I&III
   -> 37C 2h -> 90C 5min -> 4C hold