Link to Primer Sequences
18SF-Remix primer (diluted to 1x)
18S-530R primer (diluted to 10uM)
Thermal cycling conditions:
Cycle 1 95°C, 5 minutes
Cycle 2 95°C, 40 seconds
Cycle 3 50°C, 30 seconds
Cycle 4 72°C, 2 minutes
Repeat cycles 2-4 for a total of 35 cycles
Cycle 5, 72°C, 10 minutes
Cycle 6, 4°C, infinite hold
We use Dynazyme polymerase (F-552L from MJ Research), with the kit components.
The 5x buffer has 15mM MgCl2 and a mix of dNTPs at 10mM each.
The final reaction volume is 25 ul.
Volume/Reagent
19.25 ul/ddH2O
2.5 ul/Buffer (including MgCl2)
0.5 ul/dNTP mix
1.0 ul/each primer
0.5 ul/Dynazyme polymerase
1.0 ul template
After the PCR is completed, we use a 1.5% Sea Plaque gel (50110 from Cambrex) (in TAE buffer) for electrophoresis of 20ul of the PCR product. From this, we excise the band and use the Qiagen (28706 from Qiagen) gel extraction kit to recover the DNA in a volume of 30ul.
From this recovered DNA, we use a volume of 4ul in a sequencing reaction.
The sequencing reactions are as follows:
4 ul of PCR product
2 ul of 2uM primer (530R, in this case)
and 4 ul of the kit sequencing mix. We use an annealing temperature of 55°C, (vs. the 50°C recommended in the kit) and 35 cycles.
DO NOT try to sequence with the primer cocktail.