AlanFung:LabNotes/Probe/2014-1-7: Difference between revisions
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==Nanodrop== | ==Nanodrop== | ||
[[File:01072014_nanodrop.JPG|400px]] | |||
*10.7ng/ul * 50ul = 535ng | *10.7ng/ul * 50ul = 535ng | ||
*120.44nM | *120.44nM | ||
==Dilute probes to 10nM== | ==Dilute probes to 10nM== | ||
*Add 24.9ul probes to 275.1ul of EB buffer | *Add 24.9ul probes to 275.1ul of EB buffer |
Revision as of 00:42, 9 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 |
Nanodrop
- 10.7ng/ul * 50ul = 535ng
- 120.44nM
Dilute probes to 10nM
- Add 24.9ul probes to 275.1ul of EB buffer
Production PCR (8 wells)
Components | 1x rxn (ul) | 8x rxn (ul) |
10nM 1st round amplicon | 0.4 | 3.2 |
KAPA SYBR FAST MM (2X) | 50 | 400 |
100uM AP1V61U | 0.4 | 3.2 |
100uM AP2V6 | 0.4 | 3.2 |
H2O | 48.8 | 390.4 |
Total | 100 | 800 |
Realtime-PCR profile
- QPCR done on ependorf
- Left:GP1_V4 Right:GP1_V6
- 95C 30sec
- (95C 10sec → 55C 20sec → 72C 30sec) x 15 cycles
- 72C 2min
- Hold at 15C
Qiaquick Column 4 Total
- Pool 2 rxn into one column
- Elute with 30ul EB buffer
Nanodrop
- 82.7ng/ul*120ul ~9.9ug yield
Exonuclease digestion to generate SS-DNA
Components | 1x rxn (ul) |
GP1_V6 Purified amplicon (~10ug) | 120 |
10X lambda Exonuclease Buffer | 15 |
Lambda Exonuclease | 6.7 |
H2O | 8.3 |
Total | 150 |
- Incubate at 37 C for 1 hr on thermocycler
- Purify with each set in 3x QIAquick columns as described above (may use 7X volume of buffer PB for ssDNA
- Elute each column with 40 ul EB buffer (Total volume ~ 240 ul)
nanodrop
- ssDNA: 37.6ng/ul * 80ul = 3ug
Removal of amplification adapters
- In 200 ul PCR tube, add 5ul USER enzyme to 80ul probes
- Incubate at 37 C for 3 hr on thermocycler.
- Add 15 ul of the following components to the USER digested DNA tubes (10ul 10X DPNII Buffer, 5ul 100uM RE-DpnII-V6 guide oligo)
- On thermocycler, incubated at 94 C for 2 min, cool down temperature to 37C for 3 min
- Keep the tube on thermocycler.
- Add 5 ul of DpnII and mix well by pipetting up and down
- Incubate at 37 C for 2 hr.