AlanFung:LabNotes/Probe/2014-1-7: Difference between revisions

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>Alan6017518
(Created page with "==GP1 V6 Padlock Probe Production== ==Overview== *Expansion PCR *Production PCR *Exonuclease digestion to generate SS-DNA *Removal of amplification adapters *Probe purificati...")
 
>Alan6017518
Line 13: Line 13:
*(32.71ng/ul * 1*10^6 ul/L) / (47450g/mol) = 689.36nM
*(32.71ng/ul * 1*10^6 ul/L) / (47450g/mol) = 689.36nM
*I will use 100nM final concentration for the expansion PCR
*I will use 100nM final concentration for the expansion PCR
==Expansion PCR==
===GP1_V6 Expansion PCR===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="font-size:12pt" valign="bottom"
|- style="font-size:12pt;font-weight:bold" align="center"
| width="133" height="15" | Components
| width="168" height="15" | Components
| width="65" | 1x rxn (ul)
| width="138" | 1x rxn (ul)
| width="65" | Final Conc


|- style="font-size:12pt" valign="bottom"
|- style="font-size:12pt" align="center"
| height="15" | GP-1 (689nM)
|style="font-weight:bold" height="15" | GP-1 (689nM)
| align="center" | 14.51
| align="center" | 14.51
| 100nM


|- style="font-size:12pt" valign="bottom"
|- style="font-size:12pt" align="center"
| height="15" | KAPA SYBR FAST MM (2X)
|style="font-weight:bold" height="15" | KAPA SYBR FAST Master Mix (2X)
| align="center" | 50
| align="center" | 50
| 1X


|- style="font-size:12pt" valign="bottom"
|- style="font-size:12pt" align="center"
| height="15" | 100uM AP1V41U
|style="font-weight:bold" height="15" | 100uM pAP1V61U
| align="center" | 0.4
| align="center" | 0.4
| 400nM


|- style="font-size:12pt" valign="bottom"
|- style="font-size:12pt" align="center"
| height="15" | 100uM AP2V$
|style="font-weight:bold" height="15" | 100uM AP2V6
| align="center" | 0.4
| align="center" | 0.4
| 400nM


|- style="font-size:12pt" valign="bottom"
|- style="font-size:12pt" align="center"
| height="15" | H2O
|style="font-weight:bold" height="15" | H2O
| align="center" | 34.69
| align="center" | 34.69
| align="center" | &nbsp;


|- style="font-size:12pt" valign="bottom"
|- style="font-size:12pt" align="center"
| height="15" | Total
|style="font-weight:bold" height="15" | Total
| align="center" | 100
| align="center" | 100
| align="center" | &nbsp;


|}
|}

Revision as of 19:45, 7 January 2014

GP1 V6 Padlock Probe Production

Overview

  • Expansion PCR
  • Production PCR
  • Exonuclease digestion to generate SS-DNA
  • Removal of amplification adapters
  • Probe purification by PAGE size-selection

Expansion PCR

  • Probe concentration from CustomArray 32.71ng/ul volume 80uL Amount 2.62ug
  • Probe size 146bp

Convert ng/ul to nM

  • Molar Mass of ssDNA probe: 146bp * (325Da/bp) = 146bp * (325 g/mol/bp) = 47450 g/mol
  • (32.71ng/ul * 1*10^6 ul/L) / (47450g/mol) = 689.36nM
  • I will use 100nM final concentration for the expansion PCR

GP1_V6 Expansion PCR

Components 1x rxn (ul) Final Conc
GP-1 (689nM) 14.51 100nM
KAPA SYBR FAST Master Mix (2X) 50 1X
100uM pAP1V61U 0.4 400nM
100uM AP2V6 0.4 400nM
H2O 34.69  
Total 100