Restriction digestion
Master mix preparation:
Prepare a master mix of the following per sample, plus 5 to 10% extra to allow for pipetting loss.
5X R/L buffer (see page 4 for recipe) 6.0 μl
EcoR I (12 U @20 U/μl) 0.6 μl
Mse I (8 U @ 4 U/μl) 2.0 μl
Water to 10 μl
Reaction time:
Incubate for at least 1 hr. at 37°C, but not longer than 3 hrs, before proceeding with the ligation.
Adapter ligation
Master mix preparation
If 10 μl of the restriction reaction was removed for gel analysis, prepare a ligation master mix of the following, per sample, plus enough for 5-10 extra samples:
EcoRI adapter (@ 5 pMol/μl) 0.5 μl
MseI adapter (@ 50 pMol/μl) 0.5 μl
ATP (10 mM, pH 8.0) 0.5 μl
5X R/L buffer 1.0 μl
sdH2O 2.0 μl
T4 DNA ligase (0.5 Weiss U @ 1 U/μl) 0.5 μl
TOTAL 5.0 μl
Reaction time:
Incubate for at least 3 hrs. (preferably overnight) @ 37° C. After incubation, dilute each R/L mix 1:10 with sdH2O.
Preamplification
Master mix preparation
Prepare a master mix with the following amounts per sample, plus 5-10 samples extra:
10X PCR buffer 3.0 μl
dNTP mixture (2.5 mM ea.) 2.4 μl
E primer (@ 50 ng/μl ≅ 8.3μM) 1.0 μl
M primer (@ 50 ng/μl ≅ 8.3μM) 1.0 μl
Taq polymerase (@ 5 U/μl) 0.4 μl
sdH2O 19.2 μl
μl per sample 27.0 μl
Important: If pre-amplifications are to be done in 96-well PVC plates instead of polycarbonate or polypropylene plates or tubes, it may be necessary to add 2.0 μl of 10 μg/μl BSA per sample to the Taq-buffer mix, and decrease the amount of H2O to 5.76 μl per sample. DO NOT use a hot start PCR protocol, or a hot start polymerase (e.g. AmpliTaq Gold), for the pre-amplification! The adapters are non-phosphorylated, so only the top strand is ligated. The bottom strand of the adapter will separate from the rest of the template first and must be re-synthesized during the initial heating stage. This requires polymerase activity during the initial heating.
PCR program
Place reactions in the thermocycler, and run the following PCR amplification profile:
28 cycles: 15 sec @ 94°C denaturation
30 sec @ 60°C annealing
60 sec + 1sec/cycle @ 72°C extension
1 cycle: 2 min @ 72°C final extension
hold: 4°C
Final Amplification
Master mix preparation (single dye reactions)
For each E/M primer combination to be used, prepare the following master mix, per sample, in an Eppendorf tube, plus enough for 5-10 samples extra:
10X PCR buffer 2.0 μl
dNTP mixture (2.5 mM ea) 1.6 μl
IRD-labeled E-primer (@ 6 ng/μl ≅ 1μM) 0.83 μl
M-primer (@ 50 ng/μl ≅ 8.3μM) 0.6 μl
Taq polymerase (@ 5U/μl) 0.24 μl
dH2O 9.73 μl
Final volume 15.0 μl
