HinP1I NEB#R0124

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	RFLP typing used the enzyme HinP1I (NEB): 6 μl of the PCR product was added to 0.4 μl 10× NEB buffer 2, 0.3 μl HinP1I (NEB), and 3.3 μl H2O and then digested at 37°C for 4 h.	A single representative clone of each RFLP type was amplified for sequencing using the protocol above and substituting Expand high-fidelity polymerase (Roche) for Taq DNA polymerase.

Protocol tips
RFLP typing used the enzyme HinP1I (NEB): 6 μl of the PCR product was added to 0.4 μl 10× NEB buffer 2, 0.3 μl HinP1I (NEB), and 3.3 μl H2O and then digested at 37°C for 4 h.
Downstream tips
A single representative clone of each RFLP type was amplified for sequencing using the protocol above and substituting Expand high-fidelity polymerase (Roche) for Taq DNA polymerase.

Publication protocol

FTHFS genes were amplified from insect guts as described by Leaphart and Lovell (12). Primers with 5 phosphate groups were purchased from Integrated DNA Technologies. Amplification reactions for cloning contained 1
M (each) primer, 1 Failsafe premix D (Epicentre), and 0.0525 U/
l Expand high-fidelity Taq polymerase (Roche). FTHFS was amplified from Cost003 in reaction mixtures containing 1 ng/
l template and following the recommended step-down protocol (12), followed by 25 cycles at an annealing temperature of 55°C. All other termite samples contained low levels of PCR-inhibiting compounds and required further dilution; these reaction mixtures contained 0.1 ng/
l template and were amplified for an additional 5 cycles at 55°C to generate a similar final concentration of product. PCRs were purified using QIAquick PCR purification kits (Qiagen) and cloned using a GC cloning and amplification kit with the LC-Kan vector (Lucigen). Cloned PCR products were screened by restriction fragment length polymorphism (RFLP) analysis. Isolated colonies were picked and placed in 10
l 1 TE and then incubated at 95°C for 5 min. This lysate was used for amplification reactions to generate a template for RFLP analyses and sequencing. Inserts were amplified using the vector primers SL1 and SR2 (Lucigen), FailSafe premix A (Epicentre), and 0.05 U/
l Taq polymerase (NEB). The thermocycling protocol was as follows: 3 min as 95°C, 30 cycles (95°C for 30 s, 55°C for 30 s, and 72°C for 1 min 30 s), and then 10 min at 72°C. RFLP typing used the enzyme HinP1I (NEB): 6
l of the PCR product was added to 0.4
l 10 NEB buffer 2, 0.3
l HinP1I (NEB), and 3.3
l H2O and then digested at 37°C for 4 h. A single representative clone of each RFLP type was amplified for sequencing using the protocol above and substituting Expand high-fidelity polymerase (Roche) for Taq DNA polymerase

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