AlanFung:LabNotes/Probe/2013-12-11
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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
Components | 1x rxn (ul) | Final Conc |
GP-1 (689nM) | 14.51 | 100nM |
KAPA SYBR FAST Master Mix (2X) | 50 | 1X |
100uM AP1V41U | 0.4 | 400nM |
100uM AP2V4 | 0.4 | 400nM |
H2O | 34.69 | |
Total | 100 |
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 |
Realtime-PCR profile
- 95C 30sec
- (95C 10sec → 55C 45sec → 70C 45sec) x 15 cycles
- 72C 3min
- Hold at 15C
Qiaquick Column Purification
Purify with one column per reaction
- In 1.5mL tube, add 6x volume of PB buffer to each tube with PCR product and mix (600ul to 100 ul of PCR product)
- Transfer 700ul of mixture to each qiaquick column
- spin at 14,000rpm for 1". Discard supernatant
- Add 750ul wash buffer to each column
- spin at 14,000rpm for 1". Discard supernatant
- repeat wash with wash buffer
- transfer column to 1.5mL lowbind tube
- Add 50ul 55C EB buffer to each column and incubate in 37C for 5"
- Elute DNA by spinning at 14,000 rpm for 1 min.
- Pool the purified amplicon into the same tube (total volume ~100ul).
- Measure DNA concentration by Nanadrop and calculate the total yield (dsDNA).
- Dilute purified 1st round amplicon to 10nM with TE buffer.
Production PCR (24 wells)
Mix the reaction mix in 50-mL tube Mix well and aliquot 100ul of reaction mix into each well with multichannel pipettes.
GP1_V4 Production PCR
Components | 1x rxn (ul) | 24x rxn (ul) |
1st round amplicon | 0.4 | 9.6 |
KAPA SYBR FAST Master Mix (2X) | 50 | 1200 |
100uM AP1V41U | 0.4 | 9.6 |
100uM AP2V4 | 0.4 | 9.6 |
H2O | 48.8 | 1171.2 |
Total | 100 | 2400 |
GP1_V6 Production PCR
Components | 1x rxn (ul) | 24x rxn (ul) |
1st round amplicon | 0.4 | 9.6 |
KAPA SYBR FAST Master Mix (2X) | 50 | 1200 |
100uM pAP1V61U | 0.4 | 9.6 |
100uM AP2V6 | 0.4 | 9.6 |
H2O | 48.8 | 1171.2 |
Total | 100 | 2400 |
Realtime-PCR profile 95C 30sec (95C 10sec → 55C 20sec → 72C 30sec) x 15 cycles 72C 2min Hold at 15C or 4C