4.4. To the first digest mix add the following:
1.00 μl R/L buffer (5 x)
2.00 μl Enzyme (2) - AseI (10U)
7.00 μl H2O (add 10 μl / tube)
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50.00 μl Incubate at 37°C for 1 h.
4.5. Anchor (adapter) preparation: Take 25 μg of the top strand oligo (61) and 22 μg of the bottom strand oligo (62) (1 μg/μl stocks) and increase volume to 100 μl H2O. This gives a 50 pM stock of TaqI anchor.
Oligo61(T-top)/Oligo62 (T-bottom) 5’-GACGATGAGTCCTGAC
TACTCAGGACTGGC- 5’
Take 2.6 μg of the top strand oligo (41) and 2.0 μg of the bottom strand oligo (42) (1μg/μl stocks) and increase volume to 100 μl H2O. This gives a 5 pM stock of AseI anchor.
Oligo41(A-top)/Oligo42 (A-bottom) 5’-CTCGTAGACTGCGTACC
CTGACGCATGGAT- 5’
4.6. For the ligation of the anchors add the following:
1.00 μl anchor (1) (50 pM: TaqI)
1.00 μl anchor (2) (5 pM: AseI)
1.00 μl 10 mM ATP
0.50 μl 1,4 All buffer (10 x)
1.00 μl T4 DNA ligase (1U/μl)
0.50 μl H2O (add 5 μl / tube)
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55.00 μl Incubate for 2 h at 37°C.
The ligation product is termed primary template and is used directly for pre-amplification.
5. Pre-amplification of the template
10.00 μl 10 x dilution Primary template
1.00 μl Primer 1 (oligo43/A00, 100 ng/μl stock)
1.00 μl Primer 2 (oligo63/T00, 100 ng/μl stock)
5.00 μl PCR buffer (10x Superbuffer, SphaeroQ)
0.50 μl dNTPs (25 mM)
0.25 μl Taq-Pol (SuperTaq 5U/μl)
32.25 μl
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50.00 μl
Cycle: [94°C, 30 sec; 55°C, 30 sec; 72°C, 60 sec] x 25 cycles
Check the products on an agarose gel and estimate the concentration. The material should appear as a smear between 50-700 bp (see Panel II below). There should be no visible smearing above 2 kb and no residue around the well (as in Panel I below). At this stage, products of abundant transcripts may be visible as prominent bands in the smear.
Dilute the material to a concentration of about 1ng/μl (usually 20 - 50 x dilution). This material (secondary template) is used as a template in the active PCR using the AFLP protocol (see Gel Sheet below).
