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==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=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="168" height="15" | Components | width="138" | 1x rxn (ul) | width="65" | Final Conc |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | GP-1 (689nM) | align="center" | 14.51 | 100nM |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | KAPA SYBR FAST Master Mix (2X) | align="center" | 50 | 1X |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 100uM pAP1V61U | align="center" | 0.4 | 400nM |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 100uM AP2V6 | align="center" | 0.4 | 400nM |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | H2O | align="center" | 34.69 | align="center" | |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 100 | align="center" | |} ==PCR Profile== [[File:01072014_Expansion.JPG|400px]] *95C 30sec *(95C 10sec β 55C 45sec β 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== [[File:01072014_nanodrop.JPG|400px]] *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)== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" valign="bottom" | width="133" height="15" | Components | width="65" | 1x rxn (ul) | width="65" | 8x rxn (ul) |- style="font-size:12pt" valign="bottom" | height="15" | 10nM 1st round amplicon | align="center" | 0.4 | align="center" | 3.2 |- style="font-size:12pt" valign="bottom" | height="15" | KAPA SYBR FAST MM (2X) | align="center" | 50 | align="center" | 400 |- style="font-size:12pt" valign="bottom" | height="15" | 100uM AP1V61U | align="center" | 0.4 | align="center" | 3.2 |- style="font-size:12pt" valign="bottom" | height="15" | 100uM AP2V6 | align="center" | 0.4 | align="center" | 3.2 |- style="font-size:12pt" valign="bottom" | height="15" | H2O | align="center" | 48.8 | align="center" | 390.4 |- style="font-size:12pt" valign="bottom" | height="15" | Total | align="center" | 100 | align="center" | 800 |} ==Realtime-PCR profile== *QPCR done on ependorf *Left:GP1_V4 Right:GP1_V6 *95C 30sec *(95C 10sec β 55C 20sec β 72C 30sec) x 15 cycles *72C 2min *Hold at 15C [[File:01072014_Production.JPG|400px]] ==Qiaquick Column 4 Total== *Pool 2 rxn into one column *Elute with 30ul EB buffer ==Nanodrop== [[File:01072014_nanodrop_2.JPG|400px]] *82.7ng/ul*120ul ~9.9ug yield ==Exonuclease digestion to generate SS-DNA== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" valign="bottom" | width="175" height="15" | Components | width="65" | 1x rxn (ul) |- style="font-size:12pt" valign="bottom" | height="15" | GP1_V6 Purified amplicon (~10ug) | align="center" | 120 |- style="font-size:12pt" valign="bottom" | height="15" | 10X lambda Exonuclease Buffer | align="center" | 15 |- style="font-size:12pt" valign="bottom" | height="15" | Lambda Exonuclease | align="center" | 6.7 |- style="font-size:12pt" valign="bottom" | height="15" | H2O | align="center" | 8.3 |- style="font-size:12pt" valign="bottom" | height="15" | Total | align="center" | 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 [[File:01082014_nanodrop.JPG|400px]] ==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 [[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 βurea (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
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