AlanFung:Protocol/Probe Preparation: Difference between revisions

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==Samples & Materials==
==Samples & Materials==
*Jan_09_CPG_D40809 100K Oligo (10uM)
* Nanosep MF Centrifugal Devices[[http://www.vwrsp.com/catalog/product/index.cgi?catalog_number=29300-646&inE=1&highlight=29300-646&reference_type=1&partnumber=ODM02C35&sim_code=1.0]]VWR Cat. 29300-646
*Econo Taq
*22G General Use Needles[http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=305155]B-D Cat. 305155
*AP1V6 AP2V6 Primer Mix (50uM)
*Surgical Blades[http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=371610]BD Bard-Parker Cat. 371610
*RNAse free H2O
*Nanodrop [http://www.nanodrop.com/Products.aspx]Thermo Scientific Cat. ND-1000
*50X SYBG I
*Multi-Channel Reagent Reservoirs[http://www.heathrowscientific.com/catalog/product?deptId=&prodId=20621&srchitm=HEA20521C&q=HS20521C]Heathrow Scientific Cat. HS20521C
*3M NaOAC
*Chromo4 Four-Color Real-Time PCR Detection System[http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&CategoryPath=/Home/Food-Animal-Environment%20Testing/Products/Food%20Testing/Real-Time%20PCR%20Kits/iQ-Check%20%3Cem%3ESalmonella%3C-em%3E%20II%20kit/&productID=359-1590G&Country=US&vertical=FSD]Bio-Rad Cat. 359-1590G
*GlycoBlue
*Corning® 15mL PP Centrifuge Tubes[http://catalog2.corning.com/Lifesciences/en-US/Shopping/ProductDetails.aspx?productid=430791(Lifesciences)&categoryname=]Corning Cat. 430791
*100% Ethanol
*Molecular Imager Gel Doc XR+ System[http://www.bio-rad.com/prd/en/US/adirect/biorad?ts=1&cmd=BRCatgProductDetail&vertical=LSR&catID=e1a0b16a-7dcb-4feb-a363-0822c8a47bcb]Bio-Rad Cat. 170-8170
*OmniPur® Urea[http://www.emdchemicals.com/nSearch/ProductDetails.aspx?m_id=9510&c_id=1&ivs=0]EMD Chemicals Inc. Cat. 9530
*USER Enzyme[http://www.neb.com/nebecomm/products/productM5505.asp]New England Biolabs Cat. M5505L
*DPNII(5KU/ml;1KU/ml)[http://www.neb.com/nebecomm/products/productR0543.asp]New England Biolabs Cat. R0543T/R0543S
*Lambda Exonuclease[http://www.neb.com/nebecomm/products/productM0262.asp]New England Biolabs Cat. M0262L
*iQ SYBR Green Supermix[http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&CategoryPath=/Home/Life%20Science%20Research/Products/Amplification%20-%20PCR/Real-Time%20PCR%20Detection%20Systems/MyiQ%20Single-Color%20Real-Time%20PCR%20Detection%20System/&productID=170-8880&Country=US&vertical=LSR]Bio-Rad Cat. 170-8880
*Nuclease-free Water (not DEPC-treated)[https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catProductDetail&productID=AM9937&catID=603437&backButton=true]Ambion Cat. AM9937
*QIAquick PCR Purification Kit[http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx?r=1745]Qiagen Cat. 28104
*Cassettes, 1.0 mm [http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catDisplayStyle&catKey=301&filterDispName=Gel%20Casting&filterType=1&OP=filter&filter=ft_1701%2Ff_203601*&_bcs_=H4sIAAAAAAAAAMXOT0vDQBAF8KP7MRYKOUiTSexFKkWqQimCCEHvy2aaLGT%2FsDNJzLd3A5YW8e71%0AMe%2FNL6%2FEe%2FTNoJlkJmuMo9FIf2Ydc9gCTNNUGDcajr5FV2hvgQwjDATooPMW4dzOlWvycx%2F2IfRG%0AKzbeUdGx7cVqJTgOKG5KUZUV3JXVrTg6SplFx7SWtT%2FxpCKuZaZseJB7nYbIR3PhhJ9fv0zb%2BxLM%0A2LrF9FGDakxEzXA5etS22R0%2FD29J9GIo9Gp%2BVoytj3OWolecd8mzEE%2Bqp2sjbBbnAXv55L%2BQ%2FoW2%0AuaZ9A9kYSz3EAQAA]Invitrogen Cat. NC2010
*Combs, 1.0 mm 10 well[http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&messageType=catProductDetail&showAddButton=true&productID=NC3010]Invitrogen Cat. NC3010
*Cassettes, 1.5 mm [http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catDisplayStyle&catKey=301&filterDispName=Gel%20Casting&filterType=1&OP=filter&filter=ft_1701%2Ff_203601*&_bcs_=H4sIAAAAAAAAAMXOT0vDQBAF8KP7MRYKOUiTSexFKkWqQimCCEHvy2aaLGT%2FsDNJzLd3A5YW8e71%0AMe%2FNL6%2FEe%2FTNoJlkJmuMo9FIf2Ydc9gCTNNUGDcajr5FV2hvgQwjDATooPMW4dzOlWvycx%2F2IfRG%0AKzbeUdGx7cVqJTgOKG5KUZUV3JXVrTg6SplFx7SWtT%2FxpCKuZaZseJB7nYbIR3PhhJ9fv0zb%2BxLM%0A2LrF9FGDakxEzXA5etS22R0%2FD29J9GIo9Gp%2BVoytj3OWolecd8mzEE%2Bqp2sjbBbnAXv55L%2BQ%2FoW2%0AuaZ9A9kYSz3EAQAA]Invitrogen Cat. NC2015
*Combs, 1.5 mm 2D well[http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=NC3502&CID=Search-nc3502]Invitrogen Cat. NC3502
*N,N,N′,N′-Tetramethylethylenediamine[http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=T7024|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC]Sigma-Aldrich Cat. T7024
*TBE Buffer (10X)[http://nationaldiagnostics.com/product_info.php/products_id/90]National Diagnostics Cat. EC-860
*Acrylamide-Bis Solution 40%, 29:1 DNA Sequencing Grade Solution[http://www.teknova.com/product-p/a4025.htm]Teknova Cat. A4025
*Ammonium persulfate[http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=A9164|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC]Sigma-Aldrich Cat. A9164
*Ethanol 200 proof (absolute) [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=E7023|SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC]Sigma-Aldrich Cat. E7023
*GlycoBlue[http://www.ambion.com/catalog/CatNum.php?AM9515]Ambion Cat. AM9515
*10 bp DNA Ladder[http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=10821015]Invitrogen Cat. 10821-105
*Novex® TBE-Urea Sample Buffer (2X)[http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=LC6876&CID=Search-LC6876]Invitrogen Cat. LC6876
*SYBR® Gold nucleic acid gel stain *10,000X concentrate in DMSO[http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=S11494&CID=Search-S11494]Invitrogen Cat.S11494
*EconoTaq Plus 2X Master Mix[http://www.lucigen.com/catalog/index.php?cPath=50_60&pricing] Lucigen Cat. 30035-1
*3M Sodium Acetate[http://www.teknova.com/product-p/s0298.htm]Teknova Cat. S0298


==Overview==
==Overview==
*Padlock probes production (Agilent Technologies)
*PCR reaction system setup
*PCR reaction system setup
*Probe purification with ethanol precipitation
*Probe purification with ethanol precipitation
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==Procedures==
==Procedures==
==Step 1: PCR reaction system setup==
==Step 0: Padlock probe production (Agilent Technologies)==
*Synthesis of long oligonucleotides (~150nt) by inkjet printing on programmable microarray
 
==Step 1: PCR amplification reaction system setup==
*Thaw Econo Taq on ice  
*Thaw Econo Taq on ice  
*Prepare strips and tubes on cooling rack
*Prepare strips and tubes on cooling rack
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| align="center" style="background:#f0f0f0;"|'''X72'''
| align="center" style="background:#f0f0f0;"|'''X72'''
| align="center" style="background:#f0f0f0;"|'''X96'''
| align="center" style="background:#f0f0f0;"|'''X96'''
| align="center" style="background:#f0f0f0;"|'''2.0mL Tube A'''
| align="center" style="background:#f0f0f0;"|'''1.5 mL Tube'''
| align="center" style="background:#f0f0f0;"|'''2.0mLTube B'''
 
|-
|-
|Jan_09_CPG_D40809 (10uM)||align="right"|0.2uL||align="right"|4.8uL||align="right"|9.6uL||align="right"|14.4uL||align="right"|19.2uL||align="right"|2.4uL||align="right"|2.4uL||
|Jan_09_CPG_D40809 (10uM)||align="right"|0.2uL||align="right"|4.8uL||align="right"|9.6uL||align="right"|14.4uL||align="right"|19.2uL||align="right"|2.4uL
|-
|-
|Econo Taq||align="right"|50uL||align="right"|1250uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|626uL||align="right"|625uL
|Econo Taq||align="right"|50uL||align="right"|1250uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|650uL
|-
|-
|AP1V6 AP2V6 Mix (50uM)||align="right"|0.4uL||align="right"|9.6uL||align="right"|4.8uL||align="right"|4.8uL
|AP1V6 AP2V6 Mix (50uM)||align="right"|0.4uL||align="right"|9.6uL||align="right"|19.2uL||align="right"|28.8uL||align="right"|38.4uL||align="right"|4.8uL
|-
|-
|RNAse free H2O||align="right"|50uL||align="right"|1200uL||align="right"|600uL||align="right"|600uL
|RNAse free H2O||align="right"|50uL||align="right"|1200uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|600uL
|-
|-
|50X SYBG I||align="right"|0.4uL||align="right"|9.6uL||align="right"|4.8uL||align="right"|4.8uL
|50X SYBG I||align="right"|0.4uL||align="right"|9.6uL||align="right"|19.2uL||align="right"|28.8uL||align="right"|38.4uL||align="right"|4.8uL
|-
|-
|Total Volume|| align="right"| 101uL||align="right"|2424uL||align="right"|1212uL||align="right"|1212uL
|Total Volume|| align="right"| 101uL||align="right"|2424uL||align="right"|4848uL||align="right"|7272uL||align="right"|9696uL||align="right"|1212uL
|}
|}


*Mix content in 2 x 2.0mL tube vortex and spin down
*Mix content in 1.5 mL tube vortex and spin down in the order of water>SYBR Green>Primer >template>econotaq
*Aliquot 100uL using P200 pipette to 24 wells (3 strips) on cooling rack  
*Aliquot 105uL using P200 pipette to strip tubes on cooling rack  
*Seal strips with strip caps
*Seal strips with strip caps
*Place into Real Time PCR
*Place into Real Time PCR
*Perform
*Perform
94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X17 > 72C 3M > 15C Hold
94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X16 > 72C 3M > 15C Hold


==Step 2: Probe purification with ethanol precipitation==  
==Step 2: Amplicons purification with ethanol precipitation==  
*Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
*Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
{| border="1" align="center"
{| border="1" align="center"
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*Aliquot about 3750uL to 2 x 15mL tube
*Aliquot about 3750uL to 2 x 15mL tube
*Store in -80C for more than 20 mins or forever
*Store in -80C for more than 20 mins or forever
*Turn on Centrifuge fix temperature at 4C
*Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
*Centrifuge frozen 15mL tubes at 4000rpm for 15m at 4C
*Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C
*Remove
*Remove all liquid '''CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE'''
 
*Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube
 
*Centrifuge at 10,000rpm for 5min at 4C
4. Remove all liqid, you should see a blue pallette at the bottom of the tube.
*Remove all liquid, air dry sample for 5min or until samples dried out
5. Add 750 uL cold 75% Ethanol, piette well and transfer to a 1.6 mL tube.
*Resuspend the DNA with 100uL RNAse free H2O
6. Spin at 10000 rpm for 5 min at 4C.
7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 100 uL dH2O.


==Step 4: Adapter Removal using Lambda Exonuclease & Denaturing ==
Step 3: Purify the reaction with Qiaquick columns
Exonuclease treatment
1. Add 5X the amount of PB buffer into each tube and load the mixture to the column
2. Spin at top speed (14000 rpm) for 1 min
3. Empty the collection tube, add 750 uL wash buffer (PE buffer) to the column
4. Spin at top speed for 1 min
5. Empty the collection tube, spin at top speed for 1 min
6. Carefully transfer the columns to clean 1.6 mL tubes
7. Place the column/tube assemblies on bench top, wait for 2 min
8. Add 50 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min.
    NOTE: Add 100ul of ddH2O to probe using nicking enzyme
9. measure the concentration with Nanodrop.
Step 4: Adapter Removal


For Jan09 exome Sets #1-4:
*Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube
*Add in (1/10 of total volume) 6uL 10X Exo buffer
*Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR


1. To each tube, add 10ul Dpn II buffer, 36ul H2O, 4ul Dpn II(50unit/ul).
==Step 5:Column purification with Qiaquick==
2. Incubate at 37C for 1hr and 75C 10min.
Purify the reaction with Qiaquick columns
3. USER enzyme digestion: add 10ul USER enzyme, and incubate at 37C for 4hr
* Add 5X sample volume of PB buffer into each tube, mix well
4. Purified the products with Qiaquick columns, eluted with 60ul ddH2O.
* Load mixture onto column
* Centrifuge at 14,000rpm for 1min
* Discard flow through and add 750uL PE buffer to each column
* Repeat Centrifuge
* Discard flow through
* Repeat centrifuge
* Transfer columns to clean 1.5mL tubes
* Place the column/tube assemblies on bench top wait for 2min
* Add in 50uL EB buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
* Repeat centrifuge
* Measure concentration with Nanodrop


For Jan09 exome Sets #5:
==Step 6:USER enzyme digestion==
1. Add 6 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5U/ul).
*Add 40ul ddH2O, 5ul USER, 37C 1h
2. Incubate at 37C for 2 hours followed by 90C for 10 min
3. perform one round of Qiaquick column purification and elute in 50ul of H2O each
3. To each tube, add 10ul Dpn II buffer, 36ul H2O, 4ul Dpn II(50unit/ul).
4. Incubate at 37C for 1hr and 75C 10min.
5. USER enzyme digestion: add 10ul USER enzyme, and incubate at 37C for 4hr
6. Purified the products with Qiaquick columns, eluted with 60ul ddH2O.


For Probe using Nicking Enzyme digestion:
purified PCR product:        50ul
10x NEBuffer2:                10ul
H2O                          35ul
Nt.AlwI (10U/ul)              3ul
1. Incubate at 37C for 3 hours>80C 20m
2. perform one round of Qiaquick column purification and elute in 100ul of ddH2O
3. add 2ul Nb.BsrDI and incubate at 65C for 3hr>80C 20m
4. perform one round of Qiaquick column purification and elute in 61ul of ddH2O


For 18bp, 27k9bp, 27k, and 22k sets:
*Add 10ul 10x DpnII buffer, 5ul RE-DpnII-ns2 guide oligo,  
1. Add 6 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5U/ul).
94C 2min -> 37C 3min -> add 5ul DpnII -> 37C 6h -> 75C 20min -> 4c hold
2. Incubate at 37C for 2 hours followed by 90C for 10 min
Purified with Qiaquick
3. perform one round of Qiaquick column purification and elute in 50ul of H2O each
4. Add 10ul 10X DpnII buffer, 5ul 100uM of guide oligo:
    (DpnIIV4 for 27k9bp probe set, and RE-DpnII-n2s for 18bp probe set and 27k set, RE_RE_DpnII_n2s for 22k set)
5. 94C 5min -> 60C 5min -> 0.1C/sec to 37C -> 37C 1min -> add 4ul DpnII restriction enzyme -> 37C 2hr
6. Add 4ul of USER enzyme -> 37C 4hr
7. Purified the products with Qiaquick columns, eluted with 60ul ddH2O.


Incubation with USER enzyme:
*Add 5ul DpNII 10x buffer and 2 uL of USER (1K/ml)enzyme incubate for 2 hours on 37C.
 
Annealing of DpnII oligo and incubation with DpnII.
*add 6ul of 10X DpnII buffer and 2uL RE-DpnIIs guide oligo (100uM)
*Perform 94C 5m > 60C 5m > 0.1C/sec to 37C > add 4ul of DpnII restriction enzyme -> 37C 2 hours > 4C hold
*Qiaquick column purification
*Elute with 60uL EB Buffer


Step 5: Page Denaturing Gel
==Step 5: Page Denaturing Gel==
1. Prepare a 2D well denaturing gel by scraping off the gel residue outside and peel of the sticker at the back of gel
*Prepare a 2D well denaturing gel by scraping off the gel residue outside and peel of the sticker at the back of gel
2. Load 2 gels into the gel running module
*Load 2 gels into the gel running module
3. Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line
*Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line
4. Pre-run the gel for 30 min at 200V  
*Pre-run the gel for 30 min at 200V  
5. Dilute sample to no more than 40ng/uL
*Dilute sample to no more than 40ng/uL
6. Dilute TBE-Urea sample buffer (2X) to 1X in each tube.
*Dilute TBE-Urea sample buffer (2X) to 1X in each tube.
  7. For 2 gels; Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer
  For 2 gels; Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer
    For 4 gels; Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL ddH2O, 10uL 2X TBE-Urea sample buffer  
For 4 gels; Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL ddH2O, 10uL 2X TBE-Urea sample buffer  
8. Denature samples and ladder at 75C for 8 min, then quickly transfer to ice, wait for 5 mins
*Denature samples and ladder at 75C for 7 min, then quickly transfer to cooling rack, wait for 5 mins
9. Flush the wells very well with P1000 & P200 pipette to remove all urea.
*Flush the wells very well with P1000 & P200 pipette to remove all urea.
  '''NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP'''  
  '''NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP'''  
10. Load the samples into the wells, run at 200V for 30 min.  
*Load the samples into the wells, run at 200V for 30 min.  
11. Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.  
*Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.  
12. Place tray on an orbital shaker for ~5 min.
*Place tray on an orbital shaker for ~5 min.


Step 6: Purify the product from the gel
==Step 6: Purify the product from the gel==
1. 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.  
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.
2.Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and  
*Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
transfer each half to one shearing assembly.
*Spin down all tubes at 13,000rpm for 2min
*In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.
3. Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
4. 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.
5. 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.


Step 7: Precipitation
==Step 7: Precipitation==
Turn on centrifuge let tit cool down to 4C
'''Turn on centrifuge let it cool down to 4C'''
1. Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
*Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
2. Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
*Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
3. Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
*Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
4. Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with  
*Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
    (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
*Let dry in hood ~ 10 min.
5. Let dry in hood ~ 10 min.
*Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
6. Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
Store at 4C if needed, spin down before proceeding
Store at 4C if needed, spin down before proceeding


Step 8: Quantification of DNA using denaturing gel and a quantitative ladder
==Step 8: Quantification of DNA using denaturing gel and a quantitative ladder==
Combine all tubes into a 1.5mL tube
Combine all tubes into a 1.5mL tube
  1. Add 4uL of 5ng/uL 90-mer, 4ul of H2O and 8 uL of 2x TBE-Urea buffer
  1. Add 4uL of 5ng/uL 90-mer, 4ul of H2O and 8 uL of 2x TBE-Urea buffer
  2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer
  2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer
  3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to ice, wait for 1 min.
  3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min.
  4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea.
  4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea.
  5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min.  Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL.
  5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min.  Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL.
  6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.
  6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.

Latest revision as of 23:27, 17 October 2009

Probe Preparation[edit]

Samples & Materials[edit]

  • Nanosep MF Centrifugal Devices[[1]]VWR Cat. 29300-646
  • 22G General Use Needles[2]B-D Cat. 305155
  • Surgical Blades[3]BD Bard-Parker Cat. 371610
  • Nanodrop [4]Thermo Scientific Cat. ND-1000
  • Multi-Channel Reagent Reservoirs[5]Heathrow Scientific Cat. HS20521C
  • Chromo4 Four-Color Real-Time PCR Detection System[6]Bio-Rad Cat. 359-1590G
  • Corning® 15mL PP Centrifuge Tubes[7]Corning Cat. 430791
  • Molecular Imager Gel Doc XR+ System[8]Bio-Rad Cat. 170-8170
  • OmniPur® Urea[9]EMD Chemicals Inc. Cat. 9530
  • USER Enzyme[10]New England Biolabs Cat. M5505L
  • DPNII(5KU/ml;1KU/ml)[11]New England Biolabs Cat. R0543T/R0543S
  • Lambda Exonuclease[12]New England Biolabs Cat. M0262L
  • iQ SYBR Green Supermix[13]Bio-Rad Cat. 170-8880
  • Nuclease-free Water (not DEPC-treated)[14]Ambion Cat. AM9937
  • QIAquick PCR Purification Kit[15]Qiagen Cat. 28104
  • Cassettes, 1.0 mm [16]Invitrogen Cat. NC2010
  • Combs, 1.0 mm 10 well[17]Invitrogen Cat. NC3010
  • Cassettes, 1.5 mm [18]Invitrogen Cat. NC2015
  • Combs, 1.5 mm 2D well[19]Invitrogen Cat. NC3502
  • N,N,N′,N′-Tetramethylethylenediamine[20]Sigma-Aldrich Cat. T7024
  • TBE Buffer (10X)[21]National Diagnostics Cat. EC-860
  • Acrylamide-Bis Solution 40%, 29:1 DNA Sequencing Grade Solution[22]Teknova Cat. A4025
  • Ammonium persulfate[23]Sigma-Aldrich Cat. A9164
  • Ethanol 200 proof (absolute) [24]Sigma-Aldrich Cat. E7023
  • GlycoBlue[25]Ambion Cat. AM9515
  • 10 bp DNA Ladder[26]Invitrogen Cat. 10821-105
  • Novex® TBE-Urea Sample Buffer (2X)[27]Invitrogen Cat. LC6876
  • SYBR® Gold nucleic acid gel stain *10,000X concentrate in DMSO[28]Invitrogen Cat.S11494
  • EconoTaq Plus 2X Master Mix[29] Lucigen Cat. 30035-1
  • 3M Sodium Acetate[30]Teknova Cat. S0298

Overview[edit]

  • Padlock probes production (Agilent Technologies)
  • PCR reaction system setup
  • Probe purification with ethanol precipitation
  • Purify the reaction with Qiaquick columns
  • Adapter Removal
  • Page Denaturing Gel
  • Purify the product from the gel
  • Precipitation
  • Quantification of DNA using denaturing gel and a quantitative ladder

Procedures[edit]

Step 0: Padlock probe production (Agilent Technologies)[edit]

  • Synthesis of long oligonucleotides (~150nt) by inkjet printing on programmable microarray

Step 1: PCR amplification reaction system setup[edit]

  • Thaw Econo Taq on ice
  • Prepare strips and tubes on cooling rack
  • Add in Content in table to tubes accordinly
Content Volume X24 X48 X72 X96 1.5 mL Tube
Jan_09_CPG_D40809 (10uM) 0.2uL 4.8uL 9.6uL 14.4uL 19.2uL 2.4uL
Econo Taq 50uL 1250uL 2400uL 3600uL 4800uL 650uL
AP1V6 AP2V6 Mix (50uM) 0.4uL 9.6uL 19.2uL 28.8uL 38.4uL 4.8uL
RNAse free H2O 50uL 1200uL 2400uL 3600uL 4800uL 600uL
50X SYBG I 0.4uL 9.6uL 19.2uL 28.8uL 38.4uL 4.8uL
Total Volume 101uL 2424uL 4848uL 7272uL 9696uL 1212uL
  • Mix content in 1.5 mL tube vortex and spin down in the order of water>SYBR Green>Primer >template>econotaq
  • Aliquot 105uL using P200 pipette to strip tubes on cooling rack
  • Seal strips with strip caps
  • Place into Real Time PCR
  • Perform

94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X16 > 72C 3M > 15C Hold

Step 2: Amplicons purification with ethanol precipitation[edit]

  • Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content Volume Needed Volume
100% Ethanol 2.5X Sample Volume 6000uL
3M NaOAC 0.1X Sample Volume 240uL
Glycoblue 1/300X Sample Volume 8uL
Sample Sample Volume 2400uL
Total Volume 8648uL
  • Mix all content by repeat pipetting using multichannel pipette
  • Pool all 24 wells PCR product into tray using multichannel pipette(P300)
  • Mix all content by repeat pipetting using multichannel pipette
  • Aliquot about 3750uL to 2 x 15mL tube
  • Store in -80C for more than 20 mins or forever
  • Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
  • Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C
  • Remove all liquid CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE
  • Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube
  • Centrifuge at 10,000rpm for 5min at 4C
  • Remove all liquid, air dry sample for 5min or until samples dried out
  • Resuspend the DNA with 100uL RNAse free H2O

Step 4: Adapter Removal using Lambda Exonuclease & Denaturing[edit]

Exonuclease treatment

  • Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube
  • Add in (1/10 of total volume) 6uL 10X Exo buffer
  • Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR

Step 5:Column purification with Qiaquick[edit]

Purify the reaction with Qiaquick columns

  • Add 5X sample volume of PB buffer into each tube, mix well
  • Load mixture onto column
  • Centrifuge at 14,000rpm for 1min
  • Discard flow through and add 750uL PE buffer to each column
  • Repeat Centrifuge
  • Discard flow through
  • Repeat centrifuge
  • Transfer columns to clean 1.5mL tubes
  • Place the column/tube assemblies on bench top wait for 2min
  • Add in 50uL EB buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
  • Repeat centrifuge
  • Measure concentration with Nanodrop

Step 6:USER enzyme digestion[edit]

  • Add 40ul ddH2O, 5ul USER, 37C 1h


  • Add 10ul 10x DpnII buffer, 5ul RE-DpnII-ns2 guide oligo,

94C 2min -> 37C 3min -> add 5ul DpnII -> 37C 6h -> 75C 20min -> 4c hold Purified with Qiaquick

Incubation with USER enzyme:

  • Add 5ul DpNII 10x buffer and 2 uL of USER (1K/ml)enzyme incubate for 2 hours on 37C.

Annealing of DpnII oligo and incubation with DpnII.

  • add 6ul of 10X DpnII buffer and 2uL RE-DpnIIs guide oligo (100uM)
  • Perform 94C 5m > 60C 5m > 0.1C/sec to 37C > add 4ul of DpnII restriction enzyme -> 37C 2 hours > 4C hold
  • Qiaquick column purification
  • Elute with 60uL EB Buffer

Step 5: Page Denaturing Gel[edit]

  • Prepare a 2D well denaturing gel by scraping off the gel residue outside and peel of the sticker at the back of gel
  • Load 2 gels into the gel running module
  • Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line
  • Pre-run the gel for 30 min at 200V
  • Dilute sample to no more than 40ng/uL
  • Dilute TBE-Urea sample buffer (2X) to 1X in each tube.
For 2 gels; Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer
For 4 gels; Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL ddH2O, 10uL 2X TBE-Urea sample buffer 
  • Denature samples and ladder at 75C for 7 min, then quickly transfer to cooling rack, wait for 5 mins
  • Flush the wells very well with P1000 & P200 pipette to remove all urea.
NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP 
  • Load the samples into the wells, run at 200V for 30 min.
  • Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.
  • Place tray on an orbital shaker for ~5 min.

Step 6: Purify the product from the gel[edit]

  • 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 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
  • Spin down all tubes at 13,000rpm for 2min
  • In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.

Step 7: Precipitation[edit]

Turn on centrifuge let it cool down to 4C

  • Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
  • Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
  • Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
  • Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
  • Let dry in hood ~ 10 min.
  • Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.

Store at 4C if needed, spin down before proceeding

Step 8: Quantification of DNA using denaturing gel and a quantitative ladder[edit]

Combine all tubes into a 1.5mL tube

1. Add 4uL of 5ng/uL 90-mer, 4ul of H2O and 8 uL of 2x TBE-Urea buffer
2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer
3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min.
4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea.
5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min.  Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL.
6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.