<p>ABI 3130xl – Microsatellite marker protocol (updated July 2005, Fargo ND)</p><p>PRIMERS:</p><p>Microsatellite primers - modify the Forward primer sequence by adding “M13” sequence eg: 5’ - CAC GAC GTT GTA AAA CGA C + microsatellite sequence – 3’</p><p>- do not modify Reverse primer sequence </p><p>- microsatellite primers synthesized by Illumina/Invitrogen in plate format</p><p>- re-suspend F-primer (50 pmol/ul) and R-primer (100 pmol/ul) in dd water</p><p>- make working solutions of F-primer (1.0 pmol/ul) and R-primer (10.0 pmol/ul)</p><p>M13 primers</p><p>Dye Set “G5”- FAM (blue): 5’ - 6-FAM CAC GAC GTT GTA AAA CGA C – 3’ VIC (green): 5’ - VIC CAC GAC GTT GTA AAA CGA C – 3’ NED (yellow): 5’ - NED CAC GAC GTT GTA AAA CGA C – 3’ PET (red): 5’ - PET CAC GAC GTT GTA AAA CGA C – 3’ </p><p>- labeled “M13” primers synthesized by Applied Biosystems Custom Oligo Service</p><p>- re-suspend “M13” primers (200 pmol/ul) in dd water</p><p>- make working solutions (10.0 pmol/ul)</p><p>Ref: Biotechniques (2001) 31(1):25-28.</p><p>1 PCR:</p><p>Reaction: Total Volume/ Reaction: 10ul DNA: 5ul x 10ng/ul = 50 ng per reaction Cocktail: 5ul per reaction</p><p>Stock Conc. Final Conc. Vol. / reaction (ul) 10X Buffer* 1X 1.0 2.5mM dNTPs 0.125mM 0.5 50mM MgCl2* 1.5mM 0.3 (Bioline buffer) 1.0 pmol/ul F-primer 0.4 pmols 0.4 10 pmol/ul R-primer 3.0 pmols 0.3 10 pmol/ul M13 primer 3.0 pmols 0.3 5U/ul Taq (NEB or Bioline) 0.05U 0.1 water 2.4 (2.1 with Mg added)</p><p>*NEB buffer has 20mM Mg included. If Bioline buffer is used, Mg is added to final 1.5mM.</p><p>- PCR reactions of different “colors” (“M13” primers) will be pooled for ABI3130xl runs (see later)</p><p>Program:</p><p>1. 94°C – 2:00 min. 2. 94°C – 1:00 min. 3. 50°C or 60°C – 1:00 min. 4. 72°C – 1:00 min. 5. go to 2 – 40 times 6. 72°C – 5:00 min. 7. 4°C – hold</p><p>POOLING PCR REACTIONS and PREPARING SAMPLES FOR ABI RUN:</p><p>- pool PCR reactions of four different “colors” </p><p>-Aliquot 3ul of pooled reactions into a new PCR plate (ABI plate)</p><p>- Prepare appropriate amount of formamide + LIZ500 size standard mix, LIZ500 size standard : formamide = 1 : 40 (i.e., add 25ul LIZ and 975ul formamide to make 1ml mix before use)</p><p>-Aliquot 7ul for formamide/size standard mix to each sample (quick spin to remove air bubbles)</p><p>2 -Denature plate at 95C for 5 min.; place on ice for 2 min.</p><p>-Place septa on plate and fit into plate retainer</p><p>-Plate is now ready for ABI run</p><p>PLATE RECORDS:</p><p>-Prepare a “plate record” for each plate and import to ABI 3130xl or enter plate record information on computer directly</p><p>3</p>