AlanFung:LabNotes/Probe/2014-1-7: Difference between revisions
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*Run gel at 200V for 30 mins | *Run gel at 200V for 30 mins | ||
*Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min | *Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min | ||
*Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 | *Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel. | ||
*Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly. | |||
*Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. | |||
*Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 50C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. | |||
*In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm) and then transfer supernatant to 1.5mL tube. | |||
Revision as of 21:11, 16 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 |
PCR Profile
- 95C 30sec
- (95C 10sec → 55C 45sec → 72C 45sec) x 15 cycles
- 72C 3min
- Hold at 15C
qiaquick
- one column for one reaction
- 6x pb buffer
- wash twice with PE Buffer
- elute with 50ul EB Buffer
Nanodrop
- 10.7ng/ul * 50ul = 535ng
- 120.44nM
- Stored in -20C (Alan Misc. #4)
Dilute probes to 10nM
- Add 24.9ul probes to 275.1ul of EB buffer
- Stored in -20C (Alan Misc. #3)
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.
Qiaquick Column Purification
- Elute with 40ul EB Buffer
Nanodrop
- ssDNA: 43.8ng/ul *40ul ~1.75ug
2D Gel Size Selection
- I will use one 6% TBE-Urea gel 2D well to perform purification (one gel can handle 2-2.5ug)
- Mix 40ul 2X TBE-Urea sample buffer in a 1:1 ration to make the final volume to 80ul
Components | 1 lane |
10bp DNA ladder | 1 ul |
2X TBE-Urea sample buffer | 5ul |
H2O | 4ul |
Total Volume | 10ul |
- Pre-run 6%TBE –urea (TBU) gel at 250 volts for 10-15 min in 0.5X TBE buffer.
- Heat DNA and DNA ladder-loading dye mixture at 75 C for 10mins, quickly transfer to ice, wait for 1 min
- load gel with ladder and probes
- Run gel at 200V for 30 mins
- Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min
- Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel.
- Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly.
- Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
- Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 50C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
- In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm) and then transfer supernatant to 1.5mL tube.
File:ZhangLab 2 2014-01-10 15hr 20min.jpg
ETOH Precipitation
- Resuspend with 15ul ddh2O for each tube (45ul total)
- Stored in -20C (Alan Misc. #2)
Quantify probes with qubit ssDNA because glycoblue will interfere with nanodrop
- 5.88ng/ul