DNA
NaCl
BSA
ligase buffer
adaptor pairs

1. Combine per sample:

0.1 ul	10X T4 DNA ligase buffer w/ATP
0.1 ul	0.5M NaCl
0.05 ul	1mg/ml BSA
1 unit	MseI (4 units = 1ul)
5 units	EcoRI (20 units = 1ul)
1 unit	T4 DNA ligase (3 units = 1ul)

2. Bring total volume to 1 ul per sample if necessary
3. Mix thoroughly and spin
4. Store on ice--use within 2 hours

1. For each sample combine:

1 ul	10X T4 DNA ligase buffer with ATP
1 ul	0.5M NaCl
0.5ul	1 mg/ml BSA
1 ul	50uM MseI adaptor pair
1 ul	5uM EcoRI adaptor pair
1 ul	enzyme master mix

2. In each tube combine:

5.5 ul of the above
5.5 ul of DNA sample (10ug/ml)

3. Incubate at room temperature overnight or at 37^oC for 2 hours

Make TE/10 (10:1 -- H2O/TE)

1. Add 190ul of TE/10 buffer (dilute)
2. Mix
3. Store at 2-6^oC for up to 1 month (longer if colder)

PCR buffer
preselective primers
restriction-ligation rxn. (if necessary)
MgCl₂
dNTPs

1. Core mix - Combine for each sample using ABI Amplitaq and associated reagents:
(make extra if going on to step VII in near future)

15 ul

2 ul	PCR buffer w/o MgCl2
1.6 ul	MgCl2
1.6 ul	dNTPs
0.2 ul	Taq polymerase
10.8 ul	H2O
Total

2. Mix
3. Combine for each sample:

15ul Core Mix
0.5ul 20uM AFLP preselective primer F
0.5ul 20uM AFLP preselective primer R

4. For each sample combine in PCR tubes:

16ul Core Mix with primers
4ul diluted restriction-ligation reaction

5. Run the following PCR program:

Preselective Amplification
Steps	Temp. oC	Time	Cycles
1	72	2 min
2	94	20 sec	20
	56	30 sec
	72	2 min
3	72	2 min
4	60	30 min
5	4	forever

1. Prepare 50ml 1% Seakem agarose gel with 2.5ul of EtBr. Use 1X TBE.
2. Prepare sample in small tube:

8ul sample
2ul loading dye

3. Load 10ul size standard in one well. Load 10ul of samples in others
4. Run for 1.5 - 2 hours (check voltage & current)

1. Combine for each reaction product:

10ul preselective amplification reaction product
190ul TE/10 buffer

2. Mix and spin
3. Store at 2-6^oC

PCR buffer
preselective primers

1. Combine the following in a PCR reaction tube for each sample:

15ul AFLP Core mix (as prepared in step IV but use 0.1 ul of Taq instead of 0.2)
3.0ul diluted preselective amp. rxn. product
1.0ul MseI (primer-Cxx) at 5 uM
1.0ul EcoRI (dye-primer-Axx) at 1 uM

2. Run the following PCR program:

Selective Amplification
Steps	Temp. oC	Time	Cycles
1	94	2 min
2	94	20 sec	10 cycles decreasing 1oC/cycle
	66	30 sec
	72	2 min
3	94	20 sec	20 cycles
	56	30 sec
	72	2 min
4	60	30 min
5	4	forever

3. Store at 2-6oC

dNTPs:

10ul each of 100mM dNTPs
360ul H₂O

Taq: ABI Amplitaq works best

Adaptor pairs:

EcoRI 5uM--mix:

2ul 500uM EcoRI adaptor pair
198 ul H₂O

MseI 50uM--mix:

20ul 500uM MseI adaptor pair
180 ul H₂O

To make 500 uM adaptor pairs (for EcoRI or MseI):

Combine:

40ul 1000uM F adaptor
40ul 1000uM R adaptor

Heat at 95^oC for 5 minutes
Let cool to room temp. for 10 minutes
Spin for 10 seconds

BSA:

100ul 10 mg/ml BSA
900 ul H₂O

NaCl:

100ul 5M NaCl
900 ul H₂O

MK 9/03