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 |
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*(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" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="168" height="15" | Components | ||
| width=" | | width="138" | 1x rxn (ul) | ||
| width="65" | Final Conc | |||
|- style="font-size:12pt" | |- 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" | |- style="font-size:12pt" align="center" | ||
| height="15" | KAPA SYBR FAST | |style="font-weight:bold" height="15" | KAPA SYBR FAST Master Mix (2X) | ||
| align="center" | 50 | | align="center" | 50 | ||
| 1X | |||
|- style="font-size:12pt" | |- style="font-size:12pt" align="center" | ||
| height="15" | 100uM | |style="font-weight:bold" height="15" | 100uM pAP1V61U | ||
| align="center" | 0.4 | | align="center" | 0.4 | ||
| 400nM | |||
|- style="font-size:12pt" | |- style="font-size:12pt" align="center" | ||
| height="15" | 100uM | |style="font-weight:bold" height="15" | 100uM AP2V6 | ||
| align="center" | 0.4 | | align="center" | 0.4 | ||
| 400nM | |||
|- style="font-size:12pt" | |- 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" | | |||
|- style="font-size:12pt" | |- style="font-size:12pt" align="center" | ||
| height="15" | Total | |style="font-weight:bold" height="15" | Total | ||
| align="center" | 100 | | align="center" | 100 | ||
| align="center" | | |||
|} | |} |
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 |