AlanFung:LabNotes/Probe/2014-1-7: Difference between revisions
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==PCR Profile== | |||
[[File:01072014_Expansion.JPG|400px]] | |||
*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== | ==Nanodrop== | ||
[[File:01072014_nanodrop.JPG|400px]] | |||
*10.7ng/ul * 50ul = 535ng | *10.7ng/ul * 50ul = 535ng | ||
*120.44nM | *120.44nM | ||
*Stored in -20C (Alan Misc. #4) | |||
==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 | ||
*Stored in -20C (Alan Misc. #3) | |||
==Production PCR (8 wells)== | ==Production PCR (8 wells)== | ||
Line 104: | Line 120: | ||
*72C 2min | *72C 2min | ||
*Hold at 15C | *Hold at 15C | ||
[[File:01072014_Production.JPG|400px]] | |||
==Qiaquick Column 4 Total== | ==Qiaquick Column 4 Total== | ||
*Pool 2 rxn into one column | *Pool 2 rxn into one column | ||
*Elute with 30ul EB buffer | *Elute with 30ul EB buffer | ||
==Nanodrop== | ==Nanodrop== | ||
[[File:01072014_nanodrop_2.JPG|400px]] | |||
*82.7ng/ul*120ul ~9.9ug yield | *82.7ng/ul*120ul ~9.9ug yield | ||
==Exonuclease digestion to generate SS-DNA== | ==Exonuclease digestion to generate SS-DNA== | ||
{| 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> | ||
Line 152: | Line 172: | ||
*Incubate at 37 C for 2 hr. | *Incubate at 37 C for 2 hr. | ||
==Qiaquick Column Purification== | ==Qiaquick Column Purification== | ||
*Elute with 40ul EB Buffer | |||
==Nanodrop== | ==Nanodrop== | ||
*ssDNA: 43.8ng/ul *40ul ~1.75ug | |||
[[File:01102014_nano.JPG|400px]] | |||
==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 | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="font-size:12pt" valign="bottom" | |||
| width="139" height="15" | Components | |||
| width="65" | 1 lane | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | 10bp DNA ladder | |||
| 1 ul | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | 2X TBE-Urea sample buffer | |||
| 5ul | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | H2O | |||
| 4ul | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | 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|400px]] | |||
==ETOH Precipitation== | |||
*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== | |||
*5.88ng/ul *45ul = 264.6ng |
Latest revision as of 21:28, 16 January 2014
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]
- 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]
- 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
Qiaquick Column 4 Total[edit]
- Pool 2 rxn into one column
- Elute with 30ul EB buffer
Nanodrop[edit]
- 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
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
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