8. Protocol for preparing LI-COR gels (follow manufacturer’s instructions)
8.1. 6% Long Ranger gel (7M Urea/1.2x TBE):
252 g Urea
72 ml Long Ranger 50% gel solution
72 ml 10x TBE
Add distilled water to a target weigh of 675 g
Use 20 ml/gel (248 x 254 mm plates, 0.25 mm spacers). Polymerise with 150 μl 10% APS and 15 μl TEMED.
8.2. To the selective amplification product add an equal vol (10μl) of formamide loading buffer (98% formamide, 10 mM EDTA pH8.0 and 0.1% Bromophenol blue). Vortex and centrifuge for 3 minutes at 1000 rpm. Denature the samples by heating 5 minutes at 94°C. The samples are placed on ice until loaded.
8.3. Pre-warm the gel to 45°C. Load 0.5-0.8 μl sample.
9. Preparation of the IRD 700 / 800 labelled SEQUAMARK™ 10 bp ladder
29 μl Milli Q
10 μl Circumvent polymerase buffer (10x, NEB)
48 μl dNTP/ddTTP mix (720μM ddTTP; 30μM dATP; 100μM dCTP; 100μM dGTP; 33μM dTTP)
5 μl Vent (exo-)DNA polymerase (NEB)
5 μl IRD700 or IRD800 primer (2 pmol/μl)
Subdivide in 4 PCR tubes
PCR: 94ºC, 5 min.; [94ºC, 30 sec; 56ºC, 30 sec; 72ºC, 30 sec] x 35 cycles; 72ºC, 7 min
10. Selective amplification with radioactively labelled primers
20x / 50x dilution secondary template 5.00 μl
Labelled-Primer 1 (AseI +2) * 0.25 μl
Primer 2 (TaqI (+2), 50 ng/μl stock) 0.3 μl
10x PCR buffer (Superbuffer, SphaeroQ) 1.0 μl
dNTPs (5 mM) 0.40 μl
Taq-Pol (SuperTaq 5U/μl)) 0.04 μl
H2O 3.01 μl
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10.00 μl
Touch-down profile:
(94ºC, 30 sec; 65 – 56ºC (decrease 0.7ºC each cycle), 30 sec; 72ºC, 60sec) x 12
Continue with :
(94ºC, 30 sec; 56ºC, 30 sec; 72ºC, 60 sec) x 24
* Labelling of the primer: 0.05 μl γ 33P label (10μCi/μl γ 33P-ATP)
0.05 μl primer (50 ng/μl)
0.01 μl T4 Kinase (10U/μl)
0.025 μl 10x T4 Kinase buffer
0.115 μl H2O
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0.25μl
Incubate for 60 min. at 37ºC. Terminate the reaction by heating at 70ºC for 10 min.
11. Protocol for preparing cDNA/AFLP polyacrylamide gels (BRL S2 system)
11.1. New glass plates are treated with 10 M NaOH to remove all dirt. The treatment is completed when the liquid stays as a film on the glass, and does no longer break into droplets. The short glass plate is siliconized or treated with Rain-X.
11.2. Clean both glass plates tap water, brush and soap. Rinse thoroughly and dry with tissue. Make sure that the water will stay as a film on long plate; the water should pearl on the short plate. When repeated cleaning with water and soap cannot achieve film or pearls respectively, then the NaOH or Rain-X treatment should be repeated. Clean with ethanol.
11.3. Put the spacers (thickness 0.4 mm) between the clean glass plates with the cushions against the edge of the shorter glass plate. Attach the clamps. Place the comb with the teeth upward and test it is neither tight nor loose.
11.4. Seal both sides with yellow tape (Scotch electrical tape). Press the tape firmly on the glass. Seal the bottom of the plates and make straight folds around the corners.
11.5. BRL S2 sequencing gels need approximately 80 ml of 5% polyacrilamide gel solution, to which 400 μl of 10% APS (w/v) and 80μl of TEMED is added. Store monthly fresh made APS and TEMED in refrigerator. Gel solution from the refrigerator will not polymerise as fast as gel solution at room temperature. Crystallisation of Urea will re-dissolve already after slightly increasing the temperature.
11.6. Pour the 5% acrylamide gel and place the comb (shark tooth) with the teeth upwards, to create a straight `running` front. The depth of the placement of the comb will determine the sample volume that can be loaded. It is recommended to align the edge of the holes in the comb with the edge of the glass (holes outside). Check the front, because irregularities will disturb the banding pattern. Put extra clamps to squeeze the comb between the glass, because the slightest amounts of acrylamide between glass and comb will remain as rubble in the wells. Allow the gel to polymerise for at least two hours. Wrap the gel with Saran against drying in case of o/n polymerisation.
