PCR Primer Descriptions and Standard Conditions:
PCR primers were selected using the program PCR-Overlap. Gene specific primers were "5' tailed" with an M13 universal sequence to make them compatible with Applied Biosystems Dye Primer sequencing.
M13 universal forward primer is 'TGTAAAACGACGGCCAGT'.
M13 universal reverse primer is 'CAGGAAACAGCTATGACC'.
A detailed protocol for PCR and sequencing is listed below:
Gibco Life Technologies eLONGase PCR kit (Cat No. 10480-028)
140 nM primer 1 (~40 mer with universal extension - 7uM (100 ng/uL) stock concentration)
140 nM primer 2
100 uM dNTP mix (4 mM stock concentration)
0.5X eLONGase Buffer A (10X stock)
0.5X eLONGase Buffer B (10X stock)
0.04 U/uL Elongase Polymerase Enzyme (1 U/uL stock)
H20 to bring up to vol. (10 uL reaction)
2 ng/uL DNA (5 ng/uL stock)
A Master Mix for 'X' number of reactions is set up and then aliquoted into wells containing DNA and followed by the addition of sample DNA.
PCR cycling conditions:
1- 94 C for 30 sec
2- 94 C for 30 sec
3- 60 C for 30 sec
4- 68 C for 2 min
Repeat 2-4 34X
5- 68 C for 5 min
All thermocycling is performed a MJ Research Tetrad.
Following PCR all products are diluted 1:4 in H20.
1 uL of diluted PCR product was used in each of the 4 A,C,G,T reactions (dye primer sequencing).
All sequencing was done with Big Dye Primer sequencing standard reactions with no modifications to the thermocycling protocol given by ABI.
Cycle Sequencing Protocol:
1- 96 C for 10 sec
2- 55 C for 5 sec
3- 70 C for 1 min
Repeat 1-3 14X
4- 96 C for 10 sec
5- 70 C for 1 min
Repeat 4-5 14X
Soak 4 C