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AlanFung:LabNotes/Probe/2013-12-11
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==GP1 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_V4 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 AP1V41U | align="center" | 0.4 | 400nM |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 100uM AP2V4 | 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" | |} ===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" | |} ==Realtime-PCR profile== [[File:12132013_GP1_V4.JPG|300px]] [[File:12132013_GP1_V6.JPG|300px]] *Left:GP1_V4 Right:GP1_V6 *95C 30sec *(95C 10sec β 55C 45sec β 70C 45sec) x 15 cycles *72C 3min *Hold at 15C ==Qiaquick Column Purification== ===Purify with one column per reaction=== *In 1.5mL tube, add 6x volume of PB buffer to each tube with PCR product and mix (600ul to 100 ul of PCR product) *Transfer 700ul of mixture to each qiaquick column *spin at 14,000rpm for 1". Discard supernatant *Add 750ul wash buffer to each column *spin at 14,000rpm for 1". Discard supernatant *repeat wash with wash buffer *transfer column to 1.5mL lowbind tube *Add 50ul 55C EB buffer to each column and incubate in 37C for 5" *Elute DNA by spinning at 14,000 rpm for 1 min. *Pool the purified amplicon into the same tube (total volume ~100ul). *Measure DNA concentration by Nanadrop and calculate the total yield (dsDNA). *Dilute purified 1st round amplicon to 10nM with TE buffer. ===Nanodrop=== [[File:12132013_GP1_V4_ND1.JPG|300px|GP1_V4]] [[File:12132013_GP1_V6_ND1.JPG|300px|GP1_V6]] *Left:GP1_V4 Right:GP1_V6 *GP1_V4:10ng/ul, 112.56nM *GP1_V6:9.9ng/ul, 111.44nM *Keep 1ul for Gel quantification ===Dilute probes to 10nM=== *GP1_V4: Add 0.85ul 112.56nM probes to 8.75ul EBuffer *GP1_V6: Add 0.86ul 111.44nM probes to 8.74ul EBuffer ==Production PCR (24 wells)== Mix the reaction mix in 50-mL tube Mix well and aliquot 100ul of reaction mix into each well with multichannel pipettes. ===GP1_V4 Production 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="199" height="15" | Components | width="138" | 1x rxn (ul) | width="65" | 24x rxn (ul) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 10nM 1st round amplicon | align="center" | 0.4 | align="center" | 9.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | KAPA SYBR FAST Master Mix (2X) | align="center" | 50 | align="center" | 1200 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 100uM AP1V41U | align="center" | 0.4 | align="center" | 9.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 100uM AP2V4 | align="center" | 0.4 | align="center" | 9.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | H2O | align="center" | 48.8 | align="center" | 1171.2 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 100 | align="center" | 2400 |} ===GP1_V6 Production 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="199" height="15" | Components | width="138" | 1x rxn (ul) | width="65" | 24x rxn (ul) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 1st round amplicon | align="center" | 0.4 | align="center" | 9.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | KAPA SYBR FAST Master Mix (2X) | align="center" | 50 | align="center" | 1200 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 100uM pAP1V61U | align="center" | 0.4 | align="center" | 9.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 100uM AP2V6 | align="center" | 0.4 | align="center" | 9.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | H2O | align="center" | 48.8 | align="center" | 1171.2 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 100 | align="center" | 2400 |} ==Realtime-PCR profile== [[File:12132013_GP1_V4_production.JPG|300px]] [[File:12132013_GP1_V6_production.JPG|300px]] *Left:GP1_V4 Right:GP1_V6 *95C 30sec *(95C 10sec β 55C 20sec β 72C 30sec) x 15 cycles *72C 2min *Hold at 15C ==EtOH precipitation== *Transfer the amplified amplicon from 96-well plate into 50-mL disposable basins (Total volume ~9.6mL) *Aliquot 1.2 mL of amplified amplicon into 15-mL tube (4 tubes) *In each tube, add 4 ul of Glycoblue, 120 ul of 3M NaOAc, and 3 mL of 100 % EtOH (2.5 volume of 100% EtOH, 1/300 volume of Glycoblue, 0.1 volume of 3M NaOAc pH 5.2 or 5.5) *Mix well and leave at -80C for at least 30 min. *Precipitate by centrifugation at ~10,000xg for 30 min, at 4 C *Discard spnt and add 800ul of cold 75% EtOH to each tube. *Use 1 mL clean pipette tip transfer DNA pellet to 1.5 mL tube. *Rinse the pellet and spin the tube at 14,000 rpm for 5 min. *Discard spnt and air-dry the pellet for 3-5 min in the hood (PCR work station). *Resuspend DNA with 150 ul ddH2O mix well by pipetting up and down. ==Qiaquick Re-purification== *Add 6x volume of PB buffer to each tube (900ul to 150 ul of DNA) *Transfer 700 ul of the mixture to each QIAquick column (6 columns total, 3 for each set) *Spin at 14,000 rpm for 1 min. Discard the spnt. *Add 750 ul of PE buffer to each tube. *Spin at 14,000 rpm for 1 min. Discard spnt (repeat washing step with buffer PE) *Spin the empty column at 14,000 rpm for 1 min. *Transfer the column to a fresh 1.5 mL tube. *Add 50 ul of 55C EB buffer to each tube *Elute the DNA by spinning at 14,000 rpm for 1 min. *Pool the purified amplicon into the same tube (total volume ~100 ul for each set). *Measure DNA concentration by Nanadrop and calculate the total yield (ds-DNA). ==Nanodrop== [[File:12142013_GP1_V4_ND.JPG|300px]] [[File:12142013_GP1_V6_ND.JPG|300px]] *Left: GP1_V4 Right GP1_V6 *GP1_V4: 18.1ng/ul *150ul = 2715 ng *GP1_V6: 20.6ng/ul *150ul = 3090 ng ==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;font-weight:bold" align="center" | width="199" height="15" | Components | width="138" | 1x rxn (ul) | width="65" | 2x rxn (ul) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | GP1_V4 Purified amplicon (~ 15ug) | align="center" | 75 | align="center" | 150 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 10X Lambda Exonuclease Buffer | align="center" | 15 | align="center" | 30 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Lambda Exonuclease | align="center" | 10 | align="center" | 20 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | H2O | align="center" | 50 | align="center" | 100 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 150 | align="center" | 300 |- style="font-size:12pt" align="center" |style="font-weight:bold" align="center" height="15" | | align="center" | | align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | height="15" | Components | 1x rxn (ul) | 2x rxn (ul) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | GP1_V6 Purified amplicon (~ 15ug) | align="center" | 75 | align="center" | 150 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 10X Lambda Exonuclease Buffer | align="center" | 15 | align="center" | 30 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Lambda Exonuclease | align="center" | 10 | align="center" | 20 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | H2O | align="center" | 50 | align="center" | 100 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 150 | align="center" | 300 |} *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== [[File:12142013_GP1_V4_ND_SS.JPG|300px]] [[File:12142013_GP1_V6_ND_SS.JPG|300px]] *Left: GP1_V4_SS Right: GP1_V6_SS *GP1_V4_SS: 2.3 ng/ul *120ul = 276ng *GP1_V6_SS: 3.3 ng/ul *120ul = 396ng *Yield is too low, may be too much lambda exo for the reaction and lead to over digestion? *Store probes in -20C for storage and troubleshoot ==Removal of amplification adapters== *SETUP RXN *Incubate at 37 C for 3 hr on thermocycler. *Add 15 ul of the following components to the USER digested DNA tubes *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 Purification== *Purify USER/DpnII digested amplicon with 4-6 QIAquick columns as described previously (use 7X volume of buffer PB). ==Nanodrop ssDNA== *Measure DNA concentration (ssDNA) by Nanodrop and calculate total yield. ==Probe purification by PAGE size-selection == ==EtOH Precipitation== ==Qubit ssDNA==
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