Wendel Lab AFLP protocol


Department of Ecology, Evolution, and Organismal Biology
Iowa State University, Ames, IA 50011, USA

DNA isolation

DNA quality is an extremely important factor, and membrane based DNA isolation techniques seem to result in higher quality DNA than CTAB or other types of isolations. We use the DNeasy minikit from Qiagen which produces relatively clean DNA in more than sufficient amounts for AFLP analysis. The only deviation from the kit protocol is in step #4. We perform the optional 5 minute spin at this point because of the large amounts of precipitate formed upon addition of Buffer AP2. Only the supernatant is applied to the lilac shredder column (vs. applying the entire content of the tube).  

Digestion/ligation reation

Although other labs have had some success when combining digestion of genomic DNA and ligation of adapters in to one reaction, we have found this step to be problematic. Digestion and ligation reactions have been much more successful when performed in separate steps. This may be due to differences in optimal temperatures for enzyme activity between the restriction enzymes and ligase. Here's how we do it:

DIGESTION:

COMPONENT

AMOUNT (ul)

DNA

200 ng

10X RE buffer

2.0

RE #1

10 units

RE #2

10 units

dH2O

XX

TOTAL

20.0

Total reaction volume should be 20 ul. We have been using approximately 200 ng of DNA in the digestion. Adjust the amount of dH2O accordingly. Incubate at 37˚C for 3 hours. Immediately proceed to the ligation reaction.

 

Preparing the adapters

1. Eco RI adapter pair:

250 ul forward adapter @ 100 uM+ 250 ul reverse adapter @ 100 uM = 500 ul @ 50 uM

2. Mse I adapter pair:

250 ul forward adapter @ 100 uM + 250 ul reverse adapter @ 100 uM = 500 ul @ 50 uM

* Heat @ 95˚C for 5 minutes then cool SLOWLY in a styrofoam box to renature.

LIGATION:

COMPONENT

AMOUNT (ul)

10X ligase buffer

4.0 (1X)

Adapter 1 (50 uM)

1.5 (75 pmoles)

Adapter 2 (50 uM)

1.5 (75 pmoles)

T4 DNA ligase (2000 U/ul)

0.01 (20 units)

dH2O

12.99

TOTAL

20

Add 20 ul ligation mix to each tube and incubate overnight at 16˚C. Add 160 ul dH2O and invert to mix. Use this mix in the +1 pre-selective amplification.

+1 pre-selective amplification:

+1 MASTER MIX:

COMPONENT

AMOUNT (ul)

10X PCR buffer

5.0 (1X)

MgCl2 (50 mM)

1.5 (1.5 mM)

dNTP (2.5 mM)

4.0 (200 uM)

+1 primer #1 (5 uM)

8.0 (40 pmoles)

+1 primer #2 (5 uM)

8.0 (40 pmoles)

Taq polymerase (5U/ul)

0.5 (2.5 U)

dH2O

13.0

TOTAL

40.0

Add 10 ul dilute digestion/ligation mixture to 40 ul +1 master mix. The reaction conditions we use are as follows:

1. 75˚C 2 min
2. 94˚C 30 sec
3. 56˚C 30 sec
4. 75˚C 2 min
5. go to #2 19 more times
6. 60˚C 30 min
7. 4˚C hold

Dilute each reaction with 720 ul dH2O. Proceed to selective +3 amplifications.

+3 selective amplifications:

COMPONENT

AMOUNT (ul)

10X PCR buffer

2.5 (1X)

MgCl2 (50 mM)

0.75 (1.5 mM)

dNTP (2.5 mM)

3.0 (300 uM)

FAM primer (5 uM)

0.75 (~4 pmoles)

TET primer (5 uM)

0.75 (~4 pmoles)

non-labeled primer (50 uM)

0.5 (25 pmoles)

Taq polymerase (5 U/ul)

0.25 (~1 unit)

dH2O

11.5

TOTAL

20.0

NOTE: We found no difference between reactions in which individual florescent-labeled primers were used and reactions in which differentially labeled (green and blue) primers were multiplexed.

Add 5 ul dilute +1 reaction to 20 ul +3 master mix. (Note: 25 ul reaction work with great success, but when the reactions are decreased to a total of 10 ul, results were less reliable). Reaction conditions:

1. 94˚C 2 min
2. 94˚C 30 sec
3. 65˚C 30 sec (reduce by 1 C per cycle)
4. 72˚C 2 min
5. go to step #2 9 more times
6. 94˚C 30 sec
7. 56˚C 30 sec
8. 72˚C 2 min
9. go to step #6 25 more times
10. 60˚C 5 min
11. 4˚C hold

PRIMER AND ADAPTER SEQUENCES:

PRIMER/ADAPTER

5' - sequence - 3'

Eco RI forward adapter

CTC GTA TAC TGC GTA CC

Eco RI reverse adapter

AAT TGG TAC GCA GTA

Mse I forward adapter

GAC GAT GAG TCC TGA G

Mse I reverse adapter

TAC TCA GGA CTC ATC

Eco RI +1 primer

TAC TGC GTA CCA ATT C - A

Mse I +1 primer

GAC GAT GAG TCC TGA GTA A - C

Mse I +3 primer_1

GAC GAT GAG TCC TGA GTA A - CAA

Mse I +3 primer_2

GAC GAT GAG TCC TGA GTA A - CAC

Eco RI +3 primer_1

(FAM) - TAC TGC GTA CCA ATT C - AGC

Eco RI +3 primer_2

(HEX) - TAC TGC GTA CCA ATT C - ACG

Eco RI +3 primer_3

(HEX) - TAC TGC GTA CCA ATT C - AAC

Eco RI +3 primer_4

(FAM) - TAC TGC GTA CCA ATT C - ACA

 

+3 primer combinations:

 

 

Eco + AGC (FAM)

Eco + ACG (HEX)

Eco + AAC (HEX)

Eco + ACA (FAM)

 

Mse + CAA

 

I

II

 

Mse + CAC

 

III

IV

 

* All data extractions were performed using Applied Biosystems' GeneScan and Genotyper software

Protocol updated October 2005. Many thanks to Jennifer Hawkins, Ryan Percifield, and Dr. Bao Liu.