AFLP
DNA Isolation v.2
AFLP
Primer Seqs. v.1
AFLP
Primer Tables AEIM
Zon Lab Contact: David Ransom
This AFLP method is based on the work of Vos et al (1995, NAR 23, 4407-4414). It uses primers +1 base for pre-amplification and primers +3 bases for selective amplification. We have seen an average of 50 to 100 bands per reaction of which ~25 bands segregate in individual haploids. For bulk segregant analysis, it is important to run 2 wt and 2 mt pools at the same time.
0. Isolation of
Genomic DNA for AFLP See additional protocols.
I. Digestion of
Genomic DNA
1. Use PCR tubes and a thermocycler for all steps. Mix pooled DNAs together first then digest.II. Ligation of Adapters
Reagents Volumes 10 X One-Phor-All+ DNA (from 50 µl stock) EcoRI/MseI Mix H2O 2. Incubate in a thermocycler at 370C for 2 hours, 720C for 10 min, to 40C > 5 min.
1. Add the following to digested DNA:III. Preamplification Reactions
Reagents Volumes Adapter ligation solution T4 DNA ligase 2. Incubate in a thermocycler at 200C for 2 hours.
3. Dilute 1:3 by adding 100 µl of TE buffer and mix. Enough for 30 preamps or about 7500 AFLPs! Some protocols suggest a 1:10 dilution.
4. Store at -200C.
1. Assemble reactions on ice.
Reagents Volumes diluted template DNA pre-amp primer mix 10 X PCR buffer 25mM Mg Taq (1:4 dilution of 4/97) 2. PCR for 20 cycles at: 940C, 30 sec; 560C, 60 sec; 720C, 60 sec; soak at 40C.
Do not preheat samples before PCR.3. Dilute 1:30 by adding 5 µl of preamp reaction to 145 µl of TE. Make multiple tubes in a batch . Each 150 µl tube is sufficient for 30 specific amplifcations. One 96 well plate will hold 24 sets. For 4 pools the stocks should be arrayed wt1, mt1 ,wt2, mt2 to aid plate and gel loading.
4. Store at -200C.
IV. Primer
labeling (very hot)
1. Assemble in PCR tubes. Enough for 50 specific amplifications.
Reagents Volumes EcoRI primer (pick one) 5 X kinase buffer gamma-32P-ATP 6000Ci/m T4 polynucleotide kinase 2. Incubate in a thermocycler at 370C for 1 hour; 720C, 10 min; soak at 40C.
V. Selective
AFLP amplification
1. Mix 1 Add to a PCR tube. Per 5 reactions:VI. Gel Analysis
Reagents Volumes labeled EcoRI primer MseI primer Mix 2. Mix 2 Add to a 1.5 ml tube. Per 10 reactions:
Reagents Volumes X 11 ddH2O 10 X PCR buffer 25mM Mg Taq (1:3 dilution of 3/97) 3. Assemble AFLP reaction on ice then spin down. It is best to add DNAs in an array ready for the multichannel gel loader.
Reagents Volumes diluted template DNA Mix 1 (primers/dNTPs) Mix 2 (Taq/ buffer) 4. PCR cycle profile for 9600
Auto cycle: [940C, 30 sec; 650C - 0.70 per cycle, 30 sec; 720C, 60 sec] for 13 cycles Cycle: [940C, 30 sec; 560C, 30 sec; 720C, 60 sec] for 23 cycles Soak: 40C PCR cycle for the MJ takes less time.
Total time 2 hours 2 min.
1. After PCR add 20 µl of Seq Stop solution.VII. Modified Protocol for Band Recovery2. Before running on a gel, incubate in a thermocycler at 940C, 4 min; soak at 40C; place on ice.
3. Denaturing gel. Load 3 µl per lane.
Per Long Ranger Gel (200ml: JT Baker # 4730-02):
Reagents Volumes Urea 10 X TBE Longer Ranger Mix dH2O TEMED fresh 10% APS Prerun in 0.6 X TBE for about 20 min. Run in 0.6 X TBE about 2 hours at 55 watts constant power until the bromphenol blue runs off the gel.
4. Do not fix. Dry. Expose O/N or about 12 hours without screens.
1. Punch holes through autorad into gel with a needle. Cut out 4 bands from individual AFLPs.2. Add 500 ul of elution buffer and incubate at 55 C for 1 hour.
3. Spin down paper at 14K 5 min. Remove 250 ul of sup. Respin to remove all debris.
4. Add an eqal volume of EtOH and place on ice for 30 min.
5. Spin 14K for 15 min at 4 C.
6. Wash 2x with 1 ml 70% EtOH
7. Speedvac dry and resuspend in 50 ul TE.
8. Use 10 ul of DNA per 100 ul PCR with 100 pmol (~1 ug) each AFLP primer.
9. Run 1/2 of reamplified PCR product on an agarose TAE gel to cut out and subclone the band.
Elution buffer:0.5 M ammonium acetate
1 mM EDTA pH8.0
0.2% SDS
AFLP
Reagents:
Digestion
1. EcoR I/Mse I Mixture. 4 U MseI + 4 U EcoRI / µl.Ligation
Add 10 µl stock Eco RI (NEB 101CS, 100 U / µl) to 250 µl stock MseI (NEB 525L, 4 U / µl).2. 10 X Reaction Buffer. (Parmacia, One-Phor-All+)
3. Adapter/Ligation Solution. E-Adapter, 5 pmoles/ µl /M-Adapter, 50 pmoles, 0.4 mM ATP, 10 mM TrisCl 7.5, 10 mM MgOAc, 50 mM KOAc.PreamplificationAdapter Prep per tube of Adapter/Ligation solution
E-Adapter: 5 pmoles / µl
E-Adapter.1 0.32 µg / µl 5 µl
E-Adapter.2 0.34 µg / µl 5 µlH2O 44 µl
10X One-Phor-All+ buffer 6 µl
Cycle 940 C, 4 min; ramp 90 min to 450 C, 30 sec; ramp 90 min to 40 C.
M-Adapter: 50 pmoles / µl
M-Adapter.1 1.56 µg / µl 10 µl
M-Adapter.2 1.33 µg / µl 10µlH2O 34 µl
10X One-Phor-All+ buffer 6 µl
Cycle 940 C, 4 min; ramp 90 min to 450 C, 30 sec; ramp 90 min to 40 C.
Adapter/Ligation Solution: 1.44 ml (60 reactions)
E-Adapter 60 µl
M-Adapter 60 µlATP 100 mmol/l 6.0 µl
10X One-Phor-All 131 µl
H2O 1,183.0 µl
5. T4 DNA Ligase. (BRL #)
6. TE Buffer. 10 mM TrisCl 8.0, 0.1 mM EDTA.
7. PreAmp Primer Mix. The primers are at a concentration of 28.2 ng/ul each, 0.26 mM each dNTP.Specific AmplificationPreAmp Primer Mix: 1.080 ml (30 reactions), 0.26 mM each dNTP
M-Pre-N 28.2 µg 28.2 µl
E-Pre-N 28.2 µg 28.2 µldNTPs 100 mM 2.8 µl each
H2O 1012 µl
8. T4 Polynucleotide Kinase. 10 U / µl (BRL 18004-010).9. 5X Kinase Buffer. (BRL 18004-010) 350 mM TrisCl 7.6, 50 mM MgCl2, 500 mM KCl, 5 mM BMeOH.
10. EcoRI Primers. 27.8 ng/ul in H2O. See attached list of primers.
EcoRI Primer: 0.5 ml (55, 25 µl labelings, 2750 AFLP reactions)
EcoRI Primer 1 µg / µl 27.8 µl
H2O 972 µl
11. Mse I Primers. 6.7 ng/ul, 0.225 mM each dNTP.
MseI Primer Mix: 1.5 ml (330 AFLP reactions)
MseI Primer 1 µg / µl 10 µl
dNTPs 100mM 3.38 µl each
H2O 1,453 µl
12. 10X PCR Buffer w/o Mg and 25 mM MgCl2. (BMB 1699121). Reactions should be 1.5 mM MgCl2 final concentration.
