TITLE: Sanger sequencing for genes causing intellectual disability
AUTHORS: Megan McSherry, Katherine E. Masih, Filiz Basak Cengiz, Daniella Nunez, Claire Sineni, Serhat Seyhan, Guney Bademci, Mustafa Tekin.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sanger sequencing was performed on available samples from all affected family members to determine whether the potential variant in the known genes of autosomal recessive intellectual disability co-segregated with the disease phenotype. Initially we performed a touchdown PCR. The amplified products were then purified using Sephadex Clean up and were consequently sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, USA). The resulting sequencing reactions were then purified Sephadex Clean up method. Capillary sequencing was performed in a Genetic Analyzer 3130 (Applied Biosystems, Thermo Fisher Scientific, USA) and the data were  analyzed using Sequencing Analysis software Sequencer 5.0. </div></div>

[STEPS]
?. [Normalize the DNA samples.]
The DNA concentration per sample is 20 ng/μL
?. [PCR]
Calculate the number of reactions to be performed for each assay, including recommended controls.  ABCD1Component Working ConcentrationFinal  Concentration25 ul reaction210x Buffer10x1x2.5 ul3dNTPs 2 mM (each)200 uM(each)2.5 ul4MgCl250 mM1.5 uM0.75 ul5Taq 5 units/ul1 unit/ul0.2 ul6Forward Primer20 uM0.48 uM0.7 ul7Reverse Primer20 uM0.48 uM0.7 ul8DNAn/a20 ng/ul1-2 ul9H2On/an/aTo 25 ul
Use negative and positive control. Prepare excess volume to account for pipetting errors.
?. [Place the plate/tubes in a PCR instrument. Use the thermal cycling conditions specified.]
ABCD1Hot Start 94°C3 minutes1 cycle2Denaturation94°C5 sec\3Annealing65°C30 sec---2 cycles4Extension72°C30 sec/5Denaturation94°C5 sec\6Annealing63°C30 sec---2 cycles7Extension72°C30 sec/8Denaturation94°C5 sec\9Annealing61°C30 sec---2 cycles10Extension72°C30 sec/11Denaturation94°C5 sec\12Annealing59°C30 sec---2 cycles13Extension72°C30 sec/14Denaturation94°C5 sec\15Annealing57°C30 sec---2 cycles16Extension72°C30 sec/17Denaturation94°C5 sec\18Annealing55°C30 sec---32 cycles19Extension72°C45 sec/20Final Extension Hold72°C7 min1 cycle214°C∞
?. [Make two Sephadex Plates (number of wells adjusted for a number of samples to be sequenced)]
1. Add dry Sephadex G-50 (VWR cat. 95016-896) to Millipore (cat. #MACL09645) 45 ul column loader (black mold plate)2. Place a  Pall Corportation multi-well plate (VWR cat. 97052-098) on top of the black mold plate3. Carefully turn both plates over and tap the mold plate causing Sephadex to fall into wells4. Add 300 ul of dH2O to each well5. Cover plate with adhesive seal (Applied Biosystems cat. 4306311). Write date on plate6. Allow plates to hydrate for 2 hours before use. Plates can stand in room temperature for 2 days. Can be stored at 4°C for two weeks (if stored at 4°C the plate must incubate at room temperature for 2 hours before use)
?. [PCR Purification (SOPE resin [Edge Biosystems cat. 72418] , combined with Sephadex Plate) ]
Remove plate from PCR machine  Sephadex must be brought to room temperature, and adhesive seal removed Place Sephadex plate on  top of 96-well Centrifuge sephadex plate for 5 min at 850 x g  and dispose of the eluate Bring PCR products to volume of 20 ul (should be what is remaining in PCR tube following Gel Run) Add 4ul of SOPE resin directly to the PCR product mix thoroughlyby tip mix Pipette the entire SOPE/PCR reaction mixture into the center of the wells of the Sephadex plate making sure the fluid runs into column  Stack the loaded Sephadex plate on a 96-well plate (Life Technologies cat. 4346907)  Tape the plates together Centrifuge for 5 minutes at 850 x g and Retain Eluate, dispose of sephadex plate  If product will not be used immediately it can be stored at 4°C overnight or -20°C long term
?. [BigDye Sequencing Reaction (optimized for Applied Biosystems 3130)]
Calculate the number of reactions to be performed for each assay  AB1Component Volume Added2BigDye Terminator Ready Reaction Mix0.75 ul35x Sequencing Buffer2 ul4Template (10-30ng)0.75 ul5Primer (one direction) at 0.1ug/ul)0.75 ul6Deionized Water5.75 ul7Total Volume (per well)10 ul
?. [BigDye Sequencing Program]
ABC196°C1 min 296°C10 sec350°C5 sec25 cycles460°C4 min54°CHold
?. [Dye Terminator Removal (Sephadex Plate)]
Remove plate from Thermal Cycler and cover with aluminum foil Bring Sephadex plate to room temperature remove adhesive seal Place Sephadex plate on top of  96-well “waste” plate Centrifuge for 5 minutes at 850 x g and dispose of the eluate  Wash the columns        a. Add 150 ul dH20 to each well        b. Centrifuge for 5 minutes at 850 x g and dispose of eluate 6.    Pipette 10 ul of dH2O directly in sequencing reactions7.    Transfer entire sequencing reaction volume (approximately 20 ul) directly to sephadex columns (load to center of the sephadex column without touching the column. 8.    Stack the Sephadex plate on top of a 96-well ABI sequencing plate (cat4346907)9.    Tape the plates together10.  Centrifuge for 5 minutes at 850 x g and Keep Eluate11.  Pipette 20 ul of dH20 directly into any empty wells that will be included in run         a. 3130 sequencer has 16 capillaries per run (there must be liquid in each well)12.  Seal the Plate with Adhesive tape, cover with aluminum foil, and label appropriately13.  Eluate can be stored at 4°C overnight, and -20°C for two days14.  For optimal results sequence immediately or asap