GP1 V6 Padlock Probe Production[edit]
Overview[edit]
- Expansion PCR
- Production PCR
- Exonuclease digestion to generate SS-DNA
- Removal of amplification adapters
- Probe purification by PAGE size-selection
Expansion PCR[edit]
- 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[edit]
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[edit]
File:01072014 Expansion.JPG
- 95C 30sec
- (95C 10sec → 55C 45sec → 72C 45sec) x 15 cycles
- 72C 3min
- Hold at 15C
qiaquick[edit]
- one column for one reaction
- 6x pb buffer
- wash twice with PE Buffer
- elute with 50ul EB Buffer
Nanodrop[edit]
File:01072014 nanodrop.JPG
- 10.7ng/ul * 50ul = 535ng
- 120.44nM
- Stored in -20C (Alan Misc. #4)
Dilute probes to 10nM[edit]
- Add 24.9ul probes to 275.1ul of EB buffer
- Stored in -20C (Alan Misc. #3)
Production PCR (8 wells)[edit]
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[edit]
- 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
File:01072014 Production.JPG
Qiaquick Column 4 Total[edit]
- Pool 2 rxn into one column
- Elute with 30ul EB buffer
Nanodrop[edit]
File:01072014 nanodrop 2.JPG
- 82.7ng/ul*120ul ~9.9ug yield
Exonuclease digestion to generate SS-DNA[edit]
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[edit]
- ssDNA: 37.6ng/ul * 80ul = 3ug
File:01082014 nanodrop.JPG
Removal of amplification adapters[edit]
- 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[edit]
- Elute with 40ul EB Buffer
Nanodrop[edit]
- ssDNA: 43.8ng/ul *40ul ~1.75ug
File:01102014 nano.JPG
2D Gel Size Selection[edit]
- 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 3X 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
- Cut out the correct band chop the band into small slices, split into 3x 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[edit]
- Add 0.1x of 3M sodium acetate and 2.5x 100% ethanol.
- Add 1uL Glycoblue so that one can precipitate better and see the DNA.
- Freeze in -70C overnight spin at 14,000 rpm for 15 min at 4C, discard supernatant with pipette.
- Add 500 uL of 75% ethanol to wash DNA, spin at 8000 rpm for 5 min at 4C, discard supernatant with first large tip and then small tip to remove as much liquid as possible.
- Let dry in hood ~ 10 min
- Resuspend with 15ul ddh2O for each tube (45ul total)
- Stored in (Maytag -20C Alan Misc. #2)
Quantify probes with qubit ssDNA because glycoblue will interfere with nanodrop[edit]
- 5.88ng/ul *45ul = 264.6ng