AFLP Protocol v 2.2

8/01/2000 Jeremy Agresti

Enzyme Mix

 

 

 

 

Volume per reaction (μl)

 

T4 DNA Ligase Buffer (10X)

0.1

 

NaCl (5M)

0.01

 

BSA (1mg/ml)

0.05

 

Mse I enzyme (4U/μl)

0.25

1 Unit

Eco RI enzyme (20U/μl)

0.25

5 Units

T4 DNA Ligase enzyme

0.17

1 Weiss unit (67 NEB CEL units

dd H2O

0.17

 

  • Make a master mix of the enzyme mix for the number of samples you have plus 10% overage.  Mix well, and set aside on ice while you mix up the Digest-Ligation mix.

 

Digest-Ligation mix

 

 

 

Volume per reaction (μl)

 

Mse adapter (50μM)

1.0

 

Eco adapter (5μM)

1.0

 

T4 DNA Ligase buffer (10X)

1.5

 

NaCl (5M)

0.15

  50mM final conc.

BSA (1mg/ml)

0.75

 

Enzyme mix (above)

1.0

 

Genomic DNA (and dd H2O to make the full volume)

10.6

250 ng (24 ng/μl is the minimum conc. to get 250ng in 10.6 ul)

Total volume

16 μl

 

  • Make a master mix of the dig-lig mix plus 10% overage (minus DNA, which should be added directly to the reaction tube).  Aliquot 5.4 μl to each tube.
  • Mix contents of reaction tubes by flicking and centrifuge briefly.
  • Incubate at room temp. (25°C) 12-15 hours. Usually this is done overnight.  Or digest at 37°C for 2 hr.
  • After incubation, add 84μl of TLE to the dig-lig (final DNA concentration = 2.5 ng/μl.
  • Store at 4°C up to one month, and at -20° for longer than one month.

 

Pre-Amplification  PCR

 

 

 

Volume in reaction (μl)

 

H2O

9.72

 

10X PCR Buffer

2.0

 

dNTPs (2.5 mM each)

1.6

200 μm each

MgCl2

0.6

1.5 mM

Eco A primer (6μM)

1.0

0.3 μM

Mse C primer (6μM)

1.0

0.3 μM

Taq (Gibco BRL 5U/μl)

0.08

  0.016 U

Diluted diglig from above

4.0

 

Total volume

20 μl

 

Pre-amp cycling conditions

 

 

Temp (°C)

Time

 

1.   72

2 m

 

2.   94

20 s

 

3.   56

30 s

 

4.   72

2 m

 

Repeat steps 2-4 19 more times.

 

5.   60

30 m

 

6.     4

hold

 

  • Dilute pre-amp product with 180 μl of TLE.
  • Store at 4°C

 

Selective amplification PCR

 

 

 

Volume in reaction (μl)

 

H2O

3.86

 

10X PCR buffer

1.0

 

dNTPs (2.5 mM each)

0.8

 

MgCl2

0.3

 

Eco ANN primer (1μM)

0.5

 

Mse CNN primer (5μM)

0.5

 

Taq

0.04

 

Diluted pre-amp from above

3.0

 

Total volume

10 μl

 

Selective amp. Cycling conditions

 

 

Temp (°C)

Time

 

1.   94

2 m

 

2.   94

20 s

 

3.   66

30 s

 

4.   72

2 m

 

Repeat steps 2-4 9 more times -1°C annealing/cycle.

 

5.   94

20 s

 

6.   56

30 s

 

7.   72

2 m

 

Repeat steps 5-7 19 more times.

 

 8.   60

30 m

 

9.     4

Hold

 

  • Add 10 μl 98% formamide loading buffer.
  • Load 3-4 μl on a preheated 5% denaturing polyacrylamide gel.  Run for 50 min. to 70 min. at 50W constant power.