ABI 3130Xl Microsatellite Marker Protocol (Updated July 2005, Fargo ND)

ABI 3130xl – Microsatellite marker protocol (updated July 2005, Fargo ND)

PRIMERS:

Microsatellite primers - modify the Forward primer sequence by adding “M13” sequence eg: 5’ - CAC GAC GTT GTA AAA CGA C + microsatellite sequence – 3’

- do not modify Reverse primer sequence

- microsatellite primers synthesized by Illumina/Invitrogen in plate format

- re-suspend F-primer (50 pmol/ul) and R-primer (100 pmol/ul) in dd water

- make working solutions of F-primer (1.0 pmol/ul) and R-primer (10.0 pmol/ul)

M13 primers

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’

- labeled “M13” primers synthesized by Applied Biosystems Custom Oligo Service

- re-suspend “M13” primers (200 pmol/ul) in dd water

- make working solutions (10.0 pmol/ul)

Ref: Biotechniques (2001) 31(1):25-28.

1 PCR:

Reaction: Total Volume/ Reaction: 10ul DNA: 5ul x 10ng/ul = 50 ng per reaction Cocktail: 5ul per reaction

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)

*NEB buffer has 20mM Mg included. If Bioline buffer is used, Mg is added to final 1.5mM.

- PCR reactions of different “colors” (“M13” primers) will be pooled for ABI3130xl runs (see later)

Program:

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

POOLING PCR REACTIONS and PREPARING SAMPLES FOR ABI RUN:

- pool PCR reactions of four different “colors”

-Aliquot 3ul of pooled reactions into a new PCR plate (ABI plate)

- 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)

-Aliquot 7ul for formamide/size standard mix to each sample (quick spin to remove air bubbles)

2 -Denature plate at 95C for 5 min.; place on ice for 2 min.

-Place septa on plate and fit into plate retainer

-Plate is now ready for ABI run

PLATE RECORDS:

-Prepare a “plate record” for each plate and import to ABI 3130xl or enter plate record information on computer directly