MS-AFLP protocol (96 well plate)
© 2008 Christoph Grunau, Michael Blouin, Please cite: Blouin et al. "No evidence for large differences in genomic methylation between wild and hatchery steelhead (Oncorhynchus mykiss)" Can. J. Fish. Aquat. Sci. 67(2): 217?224 (2010) [REF]
This protocol was developed as part of a Master project of Virginie Thuiller. It is based on an AFLP protocol published by Hazen, S.P., Leroy, P., Ward, R. 2002. AFLP in Triticum aestivum L.: patterns of genetic diversity and genome distribution. Euphytica 125 (1): 89-102, 2002.
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prepare high-quality DNA (e.g. by Proteinase K + phenole-chloroform extraction)
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1st day:
- dilute DNA to 20 ng/µl (at least 22 µl per enzyme mix), mix well
- prepare 22 DNA samples
- NOTE: master mix are calculated with +10%
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prepare digestion master mix HpaII-EcoRI:
| 50x | 0.1 µl (1 U) | HpaII | 5.5 µl |
| 50x | 0.12 µl (1 U) | EcoRI | 6.6 µl |
| 50x | 4 µl | buffer NEB1 10x | 220 µl |
| 50x | 25.78 µl | sterile destilled water | 1418 µl |
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prepare digestion master mix MspI-EcoRI:
| 50x | 0.1 µl (2 U) | MspI | 5.5 µl |
| 50x | 0.12 µl (1 U) | EcoRI | 6.6 µl |
| 50x | 4 µl | buffer NEB2 10x | 220 µl |
| 50x | 25.78 µl | sterile destilled water | 1418 µl |
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prepare digestion master mix AciI-EcoRI:
| 50x | 0.1 µl (1 U) | AciI | 5.5 µl |
| 50x | 0.12 µl (1 U) | EcoRI | 6.6 µl |
| 50x | 4 µl | buffer NEB3 10x | 220 µl |
| 50x | 25.78 µl | sterile destilled water | 1418 µl |
- mix well
- distribute 30 µl of each master mix into 48 wells of a 96 well plate
- add 10 µl of each DNA sample to the enzyme mix, do 2 restriction reactions for each DNA sample, mix by pipetting
- as positive control add 10 µl of a DNA sample with a known profile into 2 wells
- as negative control add 10 µl of water into 2 wells (total number of samples is 24 now)
- seal with aluminum sheet, centrifuge shortly
- incubate at 37°C for 2 to 3 hours
- prepare AciI/HpaII/MspI linker/adaptators for 200 reactions:
| AciI/HpaII Linker 1 | GACGATGAGTCTAGAA | 100 µM | 100 µl |
| AciI/HpaII Linker 2 | CGTTCTAGACTCATC | 100 µM | 100 µl |
| TRIS/Cl pH 8 | | 1 M | 2 µl |
| NaCl | | 5 M | 2 µl |
| EDTA | | 0.5 M | 0.4 µl |
- prepare EcoRI linker/adaptators for 200 reactions:
| destilled sterile water | | | 175.5 µl |
| EcoRI Linker 1 | CTCGTAGACTGCGTACC | 100 µM | 10 µl |
| EcoRI Linker 2 | AATTGGTACGCAGTCTAC | 100 µM | 10 µl |
| TRIS/Cl pH 8 | | 1 M | 2 µl |
| NaCl | | 5 M | 2 µl |
| EDTA | | 0.5 M | 0.4 µl |
- heat 10 min to 65°C, cool slowly to room temperature
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prepare ligation master mix:
| | 1x | 96x (2 enzymes) | 144x (3 enzymes) |
| T4 ligase NEB | 400 U/µl | 0.5 µl (200 U) | 53 µl | 79 µl |
| ligation buffer | 10x | 1 µl | 106 µl | 158 µl |
| EcoRI Linker | 5 µM | 1 µl | 106 µl | 158 µl |
| AciI/HpaII Linker | 50 µM | 1 µl | 106 µl | 158 µl |
| ATP | 10 mM | 2 µl | 192 µl | 317 µl |
| sterile destilled water | | 4.5 µl | 475 µl | 648 µl |
- mix well
- inactivate digestion by incubation at 65°C for 20 min
- add 10 µl of ligation master mix to each well, mix by pipetting
- seal with aluminum sheet, centrifuge shortly
- incubate at 37°C for 2 to 3 hours
- prepare PCR master mix:
| | 1x | 96x (2 enzymes) | 144x (3 enzymes) |
| Taq 10x buffer | high efficiency | 2 µl | 211 µl | 317 µl |
| Taq polymerase | 0.5 U/µl | 2 µl | 211 µl | 317 µl |
| dNTPs | 10 mM each | 2 µl | 211 µl | 317 µl |
primer AciI/HpaII
GATGAGTCTAGAACGGTCC | 10 µM | 2 µl | 211 µl | 317 µl |
fluorescent primer EcoRI
GACTGCGTACCAATTCCTG | 0.5 µM | 2 µl | 211 µl | 317 µl |
- mix well
- distribute 10 µl to one or two 96 well PCR plates
- dilute 10 µl of digestion/ligation reaction with 190 µl sterile destilled water (1:20)
- Mix well by pipetting through about 100 µl pipettes
- add 10 µl of this diluted DNA to the 10 µl PCR master mix
or:
- add 10 µl of undiluted DNA to the PCR master mix and use PCR conditions II
- seal with aluminum sheet, centrifuge shortly
- PCR:
| 96°C | 30 sec |
| 14 cycles: | |
| | 96°C | 30 sec |
| | 65°C | 30 sec |
| | decrease temperature by 0.7°C every cycle |
| | 72°C | 1 min |
| 25 cycles (15 cycles for PCR condition II): | |
| | 96°C | 30 sec |
| | 56°C | 30 sec |
| | 72°C | 1 min |
| 60°C | 30 min |
| 4°C | forever |
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2nd day:
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prepare master mix for CEQ-8000 capillary sequencer:
| 1x | 96x (2 enzymes) | 144x (3 enzymes) |
| SLS | 40 µl | 4032 µl | 6336 µl |
| 600 bp size marker | 0.1875 µl | 20 µl | 29 µl |
- distribute 40 µl master mix into 96 well plates
- add 1 µl of MS-AFLP PCR to each well
- add one drop of mineral oil to each well
- seal with aluminum sheet, centrifuge shortly
- run on CEQ-8000
- call binary data
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