AlanFung:LabNotes/Probe/2013-12-11: Difference between revisions
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===GP1_V4 Expansion PCR=== | ===GP1_V4 Expansion PCR=== | ||
===GP1_V6 Expansion PCR=== | ===GP1_V6 Expansion PCR=== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="font-size:12pt;font-weight:bold" align="center" | |||
| width="168" height="15" | Components | |||
| width="138" | 1x rxn (ul) | |||
| width="65" | Final Conc | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | GP-1 (689nM) | |||
| align="center" | 14.51 | |||
| 100nM | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | KAPA SYBR FAST Master Mix (2X) | |||
| align="center" | 50 | |||
| 1X | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | 100uM pAP1V61U | |||
| align="center" | 0.4 | |||
| 400nM | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | 100uM AP2V6 | |||
| align="center" | 0.4 | |||
| 400nM | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | H2O | |||
| align="center" | 34.69 | |||
| align="center" | | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | Total | |||
| align="center" | 100 | |||
| align="center" | | |||
|} |
Revision as of 00:34, 12 December 2013
GP1 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_V4 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 |