4. Repeat incubation at 94
C for 1 min, 37
C for 1 min, and
72
C for 2 min and a final step at 72
C for 5 min to indicate
that all the final amplification products are of full length.
5. Mix amplified DNA samples (20 μl) with 2 μl loading buffer
(0.1% bromophenol blue, 0.25% xylene cyanol, 40% w/v
sucrose in water) and run on a 1.6% (w/v) agarose gel containing 1 μg/ml ethidium bromide at room temperature and 60 V
for 3 h in TBE running buffer.
6. Visualize gels with amplification fragments and photograph
under UV light.
7. Use 100 bp DNA marker as a reference.
Table 1
Nucleotide sequences of RAPD primers
S. No.
Oligonucleotide primer
Sequences (5
0 –3
0 )
1
OPAX1
GTGTGCCGTT
2
OPAX13
CAGCAATCCC
3
OPAX15
AGTGCACACC
4
OPAX17
TGGGCTCTGG
5
OPAD2
CTGAACCGCT
6
OPAD4
GTAGGCCTCA
7
OPAD5
ACCGCATGGG
8
OPAD6
AAGTGCACGG
9
OPAD9
TCGCTTCTCC
10
OPAD12
AAGAGGGCGT
11
OPAD14
GAACGAGGGT
12
OPAV3
TGTAGCCGTG
13
OPAV10
GGACCTGCTG
14
OPAV11
GACCCCGACA
15
OPAV20
TCATGCGGCA
16
OPAX15
AGTGCACACC
17
OPAX17
TGGGCTCTGG
18
OPAX13
CAGCAATCCC
19
OPBD01
GAGCCCCGAA
20
OPBD12
GGGAACCGTC
21
OPBD13
CCTGGACGGC
92
Laboratory Techniques for Entomopathogenic Nematodes