AlanFung:Protocol/Probe Preparation: Difference between revisions

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==Samples & Materials==
==Samples & Materials==
*iQ SYBR Green Supermix [[Bio-Rad Cat. 170-8880]]
* 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
*Nuclease-free Water (not DEPC-treated) [[Ambion Cat. AM9937]]
*22G General Use Needles[http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=305155]B-D Cat. 305155
*QIAquick PCR Purification Kit [[Qiagen Cat. 28104]]
*Surgical Blades[http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=371610]BD Bard-Parker Cat. 371610
*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]]
*Nanodrop [http://www.nanodrop.com/Products.aspx]Thermo Scientific Cat. ND-1000
*Cassettes, 1.5 mm [[Invitrogen Cat. NC2015]]
*Multi-Channel Reagent Reservoirs[http://www.heathrowscientific.com/catalog/product?deptId=&prodId=20621&srchitm=HEA20521C&q=HS20521C]Heathrow Scientific Cat. HS20521C
*Template (10uM)
*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
*EconoTaq Plus 2X Master Mix [[Lucigen Cat. 30035-1]]
*Corning® 15mL PP Centrifuge Tubes[http://catalog2.corning.com/Lifesciences/en-US/Shopping/ProductDetails.aspx?productid=430791(Lifesciences)&categoryname=]Corning Cat. 430791
*Primer (F)
*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
*Primer (R)
*OmniPur® Urea[http://www.emdchemicals.com/nSearch/ProductDetails.aspx?m_id=9510&c_id=1&ivs=0]EMD Chemicals Inc. Cat. 9530
*Nuclease-free Water (not DEPC-treated) [[Ambion Cat. AM9937]]
*USER Enzyme[http://www.neb.com/nebecomm/products/productM5505.asp]New England Biolabs Cat. M5505L
*50X SYBG I
*DPNII(5KU/ml;1KU/ml)[http://www.neb.com/nebecomm/products/productR0543.asp]New England Biolabs Cat. R0543T/R0543S
*3M NaOAC
*Lambda Exonuclease[http://www.neb.com/nebecomm/products/productM0262.asp]New England Biolabs Cat. M0262L
*GlycoBlue
*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
*100% Ethanol
*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
*75% Ethanol
*QIAquick PCR Purification Kit[http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx?r=1745]Qiagen Cat. 28104
*QIAquick PCR Purification Kit [[Qiagen Cat.28104 & 28106]]
*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
*Buffer PB [[Qiagen Cat. 19066]]
*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
*QIAquick Spin Column
*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
*Buffer PE [[Qiagen Cat. 19065]]
*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
*Nanodrop
*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
*10X EXO Buffer
*TBE Buffer (10X)[http://nationaldiagnostics.com/product_info.php/products_id/90]National Diagnostics Cat. EC-860
*Lambda Exonuclease
*Acrylamide-Bis Solution 40%, 29:1 DNA Sequencing Grade Solution[http://www.teknova.com/product-p/a4025.htm]Teknova Cat. A4025
*USER Enzyme [[NEB Cat. M5505L]]
*Ammonium persulfate[http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=A9164|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC]Sigma-Aldrich Cat. A9164
*DpnII [[NEB Cat. R0543M]]
*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
*10X DpnII buffer [[NEB Cat.  
*GlycoBlue[http://www.ambion.com/catalog/CatNum.php?AM9515]Ambion Cat. AM9515
*RE-DpnIIs guide oligo
*10 bp DNA Ladder[http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=10821015]Invitrogen Cat. 10821-105
*2D Gel
*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
*10X TBE buffer
*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
*0.5X TBE buffer
*EconoTaq Plus 2X Master Mix[http://www.lucigen.com/catalog/index.php?cPath=50_60&pricing] Lucigen Cat. 30035-1
*orbital shaker
*3M Sodium Acetate[http://www.teknova.com/product-p/s0298.htm]Teknova Cat. S0298
*10bp Ladder
*SYBR Gold


==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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94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X16 > 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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*Resuspend the DNA with 100uL RNAse free H2O
*Resuspend the DNA with 100uL RNAse free H2O


 
==Step 4: Adapter Removal using Lambda Exonuclease & Denaturing ==
 
==Step 4: Adapter Removal==
Exonuclease treatment
Exonuclease treatment


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*Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR
*Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR


 
==Step 5:Column purification with Qiaquick==
 
Purify the reaction with Qiaquick columns
Purify the reaction with Quaquick columns
* Add 5X sample volume of PB buffer into each tube, mix well
* Add 5X sample volume of PB buffer into each tube, mix well
* Load mixture onto column
* Load mixture onto column
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* Measure concentration with Nanodrop  
* Measure concentration with Nanodrop  


==Step 6:USER enzyme digestion==
*Add 40ul ddH2O, 5ul USER, 37C 1h
*Add 40ul ddH2O, 5ul USER, 37C 1h



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.