John M. S. Bartlett.pdf - Bio-Nica.info

Detection of MIS 303
Table 1
Sequence of the Forward and Reverse Primers for Each Microsatellite
and the Annealing Temperature Used in the PCR
Primer name Primer sequence Annealing temp
D9S126
Forward ATT GAA ACT CTG CTG AAT TTT CTG 50°C
Reverse
CAA CTC CTC TTG GGA ACT GC
D9S259
Forward GGC ATC ATT GCA CCA T 50°C
Reverse
GGA TGG ATC TTA TGG GTG GAA
D9S275
Forward CAG GAA CTT GTC CAT TCT C 47°C
Reverse
TCT ATT ATT GCC TTA CTC ACA G
D9S195
Forward AGC TCA GCA CGG AGG G 53°C
Reverse
AGG GCA GGT TCC TAC AAA
D9S258
Forward GCT AGA GAT GCC CTTCGAG TG 53°C
Reverse
AGG ATT TAT AGA AAG TCC AAA ACC C
D9S102
Forward ATA GAC TTC CAG ACA GAT AG 50°C
Reverse
CCT CTC TCA TTC CTG GTA CT
GSN
Forward CAG CCA GCT TTG GAG ACA AC 50°C
Reverse
TCG CAA GCA TAT GAC TGT AA
D9S1199
Forward AAA AAT CAT GTG CAT CAA TTC C 53°C
Reverse
CCA GAG AAG CAG AAC CAA CG
D9S1198
Forward TGG GAG AGG GAA AAT GCT ATC 47°C
Reverse
GTA CTC CAG CCT GGG TGG
2. Materials
1. Xylene (BDH, UK).
2. 100%, 95% and 70% alcohol (BDH, UK).
3. 0.05% Toluidine blue dissolved in distilled water (Sigma Aldrich, UK).
4. Leitz microscope and Leica micromanipulator (Leica, UK).
5. Microdissecting needle and needle holder (Leica, UK).
6. Protein digestion buffer (0.5% Tween in 50 mM Tris-EDTA, pH 8.5).
7. 0.5 mg/mL proteinase K, add 450 µL of protein digestion buffer to 50 µL of 10 mg/m
proteinase K dissolved in water (Sigma Aldrich, UK).
8. 37°C water bath.
9. MJ Research PTC-225 Peltier Thermal Cycler (Genetic Research Instrumentation, UK).
10. 2 µM forward and reverse fluorescent-labeled primers per reaction (MWG-Biotech, UK
Ltd). Primers are present in the reaction at 0.2 µM. For individual primer sequences,
refer to Table 1.