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

324 Stirling
9. TAE (20×): 484 g of Tris, 114 mL of glacial acetic acid, 20 mL of 0.5 M EDTA. Dissolve
in 3 L of distilled water then make up to 5 L with distilled water.
10. TAE (1×): 120 dilution of 20× TAE in distilled water.
11. 8% polyacrylamide gel.
12. dNTPs: supplied separately as four different types at concentrations of 100 mM. For use
at 5 mM, take 100 µL of each stock dNTP and add to one tube 7600 µL of sterile distilled
water. Aliquot and store at –20°C.
Oligo F2 Wild
5′-CAC TGG GAG CAT TGA GGA TC-3′
Oligo F2 Mutant
5′-CAC TGG GAG CAT TGA GGA TT-3′
Oligo F2 Consensus 5′-TCT AGA AAC AGT TGC CTG GC-3′
Oligo F5 Wild
5′-CAG ATC CCT GGA CAG ACG-3′
Oligo F5 Mutant
5′-CAG ATC CCT GGA CAG ACA-3′
Oligo F5 Consensus 5′-TGT TAT CAC ACT GGT GCT TAA-3′
Oligo F9 Forward
5′-CTC CTG CAG CAT TGA GGG AGA TGG ACA TT-3′
Oligo F9 Reverse
5′-CTC GAA TTC GGC AAG CAT ACT CAA TGT AT-3′
13. Oligonucleotides. The oligonucleotides must be diluted to 100 pmol/µL on arrival. Aliquot
in 20-µL volumes and store at –20°C.
3. Methods
1. Using sterile pipet tips, take 1.0 µL of each sample into identified wells in plate, one
labeled ‘W’ (wild type), the other ‘M’ (mutant). Ensure that the DNA sample is pipetted
directly into the bottom of the respective well.
2. Prepare PCR mastermixes W and M with the following: DNTP (n × 1.5 µL); 10× polymerase
reaction buffer (n × 2.5 µL); oligonucleotides (each (n × 0.5 µL); MgCl 2 (25 mM;
(n × 2 µL); Taq polymerase (n × 0.2 µL); Sterile distilled water (n × 15.4 µL); where
n = number of samples/controls + 2.
3. Mix mastermixes W and M and add 24 µL to each corresponding well. Cover and seal
tightly with adhesive film and mix well but carefully by agitation on a plate shaker.
4. Place plate into thermal cycler and run the following program: 94°C for 5 min (94°C for 15 s,
55°C for 15 s, 72°C for 30 s × 35 cycles), and 72°C for 10 min.
5. When cycles are complete, add 4 µL of loading buffer to each sample well and agitate
to mix.
6. Load samples (approx 20 µL) into the wells of an 8% polyacrylamide gel in the vertical
electrophoresis unit and electrophorese at 250 volts for approx 1.25 to 1.5 h along with a
strategically placed molecular weight marker.
7. Remove gel from tank and stain for 2 to 5 min in discarded buffer from upper tank
containing 20 µL of ethidium bromide solution.
8. Interpretation.
The amplification generates three fragments: Prothrombin (F.II), 340 bp; Factor IX
(control), 250 bp; and Factor V, 174 bp. Each set of two tubes, wild type and mutant,
will show bands in the pattern below depending upon the allele detected.
Wild type
Mutant
Homozygous negative –
Heterozygous – –
Homozygous positive –
The Factor IX control fragments must be present in both the wild and mutant tubes
to interpret the P20210A and V Leiden status. Specimens should be reanalyzed if be