Primers and Protocols


Currently I am developing primers for nuclear protein-coding genes for amplification across Hymenoptera. Some of these primers are useful for higher level phylogenetic analyses, while others are being developed for species level relationships. Preliminary results show that the species level genes work as independent tests against species relationships determined with the barcoding region of CO1. Please contact me for more information.

CAD CPSase small chain
Finally got Evaniids, Ichoids are good, now on to the rest!

Protocols: For Evanioidea:

Normally I do a nested PCR using 54F/405R in the first PCR followed by 314F/710R. The first primers will amplify about a 1400-1500 amplicon in Evaniids (800-1000 in Braconids, 1000-1200 in Ichneumonids). The 314-710 is about 500-550 bp long (depending on intron length) and therefore much more useful for sequencing for Evaniids.

Ichneumonoidea can be sequenced directly with the Moutlon and Wiegmann (2004) primers without nested re-amplification.

All primers listed 5 to 3.

These are the primers of Moulton and Wiegmann (2004) (see CAD map in their paper)
CAD54F:
GTNGTNTTYCARACNGGNATGGT
CAD405R:
GCNGTRTGYTCNGGRTGRAAYTG

These are my primers:
CAD314F:
GYG ANA CNC CNA GYC AYT G
CAD710R:
GGT CCR TTR CTN ARR AAN AAN C

Using the following concentrations and protocols: (all in degrees Celsius):

Initial denaturation: 94 deg - 4 minutes
Then 3 cycles of:
94 deg - 30sec
59 deg - 30sec
72 deg - 90sec

Then 5 cycles of
94 deg - 30sec
57.5 deg - 30 sec
72 deg - 90 sec

Then 28 cycles of:
94 deg - 30 sec
54 deg - 30 sec
72 deg - 90 sec

Then final extension
72 deg - 3 min

This reaction seems sensitive to reagents used, I use:

25uL reactions with 200uM dNTP's, 1X buffer (standard buffer from NEB that comes with the polymerase, contains 1.5mM Mg++), 4mM MgSo4, 0.4uM each primer; 1 unit of Taq (standard taq polymerase from New England Biolabs), and 1 - 1.5uL template DNA.


Other Nuclear Genes
Please contact me for primers and protocols

Alpha mannosidase II (CG18802)
Tested successfully on: Braconidae, Ichneumonidae, Evaniidae, Gasteruptiidae, Aulacidae, Megaspilidae, Tenthredinidae, Ptermomalidae, Apidae

Dopa decarboxylase (CG10697)
Tested successfully on: Ichneumonidae, Ptermomalidae, Apidae

CG1475 (RPL13a)
Tested successfully on: Braconidae, Ichneumonidae, Evaniidae, Gasteruptiidae, Aulacidae, Tenthredinidae, Ptermomalidae, Apidae

CG7808 (RPL15)
Tested successfully on: Braconidae, Ichneumonidae, Evaniidae, Gasteruptiidae, Aulacidae, Tenthredinidae, Ptermomalidae, Apidae

CG17420 (RPS8)
Tested successfully on: Braconidae, Evaniidae, Tenthredinidae, Ptermomalidae, Apidae

CG17420 (RPS8)
Tested successfully on: Braconidae, Evaniidae, Tenthredinidae, Ptermomalidae, Apidae

CG8415 (RPS23)
Tested successfully on: Braconidae, Ichneumonidae, Evaniidae, Gasteruptiidae, Aulacidae, Megaspilidae, Ceraphronidae, Tenthredinidae, Ptermomalidae, Apidae

CG7283 (RPL10ab)
Tested successfully on: Ichneumonidae, Tenthredinidae, Ptermomalidae, Apidae

CG7424 (RPL36A)
Tested successfully on: Tenthredinidae, Ptermomalidae, Apidae



For Ribosomal proteins, all genes span at least one intron. Exons useful for higher level phylogenetics, introns useful for species and population level analyses.
More coming soon!