PROTOCOLS FOR

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PROTOCOLS FOR Powered By Docstoc
					                       PROTOCOLS FOR
       AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS (AFLPS)

These are the primary steps for conducting AFLPs adapted for an automated sequencer.
1. Sample preparation—p. 2
    a. Extract genomic DNA
    b. Clean DNA
2. Quantification of DNA concentration—p. 2
    a. Calibrate machine
    b. Quantify samples
    c. Cleanup
3. Restriction enzyme digestion--optional for weak DNA samples—p. 3
    a. Digest DNA with MseI and EcoRI
    b. Verify successful digestion
4. Adjust DNA concentration—p. 3
5. Restriction-ligation reaction—p. 4
    a. Prepare enzyme master mix
    b. Anneal adaptor pairs
    c. Prepare restriction-ligation master mix
    d. Complete restriction-ligation
6. Pre-selective amplification—p. 5
    a. Prepare pre-selective amplification master mix
    b. Complete amplification
    c. Confirm successful amplification
7. Selective amplification—p. 5-6
    a. Prepare template
    b. Prepare selective amplification master mix
    c. Complete amplification
8. Sequence analysis preparation—p. 6
9. Appendix I: Supplies for AFLPs—p. 7
10. Appendix II: AFLP Recipes—p. 8

Note
Available kits lack three solutions which we mix up ourselves; see Appendix II: AFLP
Recipes:
   a) NaCl (0.5M)
   b) 1X TE0.1 buffer
   c) BSA (1 mg/ml)
                                                                                            2


1--Sample Preparation
EXTRACT GENOMIC DNA
We use a Promega Wizard Genomic DNA Purification kit (catalog # A1125). Other
methods are fine, as long as very clean DNA results. If necessary, another kit (such as
Qiagen’s DNEasy kit) may be necessary for samples with abundant contaminants that
inhibit restriction digestion or PCR.


2--Quantification of DNA Concentration
This procedure uses Dr. Vis's GeneQuant machine. Make arrangements ahead of time to
visit her lab and use the machine.
CALIBRATE MACHINE
1. For each sample, pipette 95 l water into labeled 1.5 ml microfuge tube; add 5 l
    sample
2. Turn on GeneQuant machine [button on back]
3. Remove cuvette from drawer below machine, carefully wipe clean with Kimwipe
4. Push "set-up" button, then push "enter" 8 times to get "set-up factor ds DNA?" on
    screen
5. Pipette 100 l water into cuvette for use as reference standard
6. Place cuvette, oriented with window front-to-back, in top of slot (don't push all the
    way down yet), then push "set ref" button
7. When machine beeps, quickly push cuvette all the way down
8. When machine beeps again, quickly pull cuvette out
9. Pipette water out of cuvette into a waste beaker

QUANTIFY SAMPLES
1. Add 100 l sample to cuvette gently, making sure no bubbles appear in window
2. Place cuvette in top of slot
3. Push "sample" button, then when machine beeps, push cuvette all the way down;
   when machine beeps again, pull out cuvette
4. If concentration for sample is not in g/l, push "conc" button once, then push
   "select" button until proper format comes up on screen
5. Record this value for genomic DNA concentration
6. Pipette out sample into waste beaker
7. Add few drops distilled water into cuvette to rinse it, then pipette it out into waste
   beaker
8. Repeat steps 1-3 and 5

CLEANUP
1. Turn off machine
2. Squirt weak acid into cuvette, pipette out into waste beaker; rinse with water and
   pipette out into waste beaker
3. Wipe outside of cuvette with Kimwipe, store in box and return box to drawer beneath
   machine
4. Remove bottles to their proper places
5. Empty out and wash the waste beaker
                                                                                             3


3--Restriction Enzyme Digestion
This step is optional with weak DNA samples, if you are certain they will digest properly.
DIGEST DNA WITH MSEI AND ECORI
1. Pipette sufficient sample for 1 g DNA into PCR tubes
2. Make master mix, including for each sample:
   a. 0.01 l MseI
   b. 0.05 l EcoRI
   c. 1 l MseI buffer
   d. 1 l EcoRI buffer
   e. 8 l water
3. Pipette 10 l master mix into each tube; fasten strip lid firmly
4. Put in thermal cycler and run "aflp-digest" program (ca. 2 hours)

VERIFY SUCCESSFUL DIGESTION
1. Prepare 1.3% agarose gel with Ethidium bromide included
2. Pipette the 10 l digested sample into wells, leaving 1-2 leftmost wells empty
3. Pipette 10 l DNA ladder/loading dye solution into leftmost well
4. Electrophorese at ca. 40 milli-Amps for 15-20 min
5. Visualize on UV transilluminator; smear of fragments ca. 100-1500 bp long indicates
   successful digestion of sample


4--Adjust DNA Concentration
1. Set aside samples with 0.091 g/l DNA concentration [= 0.5 g DNA in 5.5 l
   water]
2. For samples with DNA concentration < 0.091 g/l:
   a. Divide 0.5 by DNA concentration
   b. If number is less than the amount of your remaining sample, pipette that number
        of microliters into a labeled 1.5 ml microfuge tube (distinguish this from rest of
        sample)
   c. If number is more than the amount of your remaining sample, simply use all of
        your sample, in the existing tube
   d. Turn warming oven on, place samples on bottom shelf, and leave for several
        hours or overnight to dry off water; check every hour or two
   e. Pipette 5.5 l thawed distilled water into each tube and vortex well
3. For samples with DNA concentration > 0.091 g/l:
   a. Pipette sufficient water into tube to lower concentration
                                                                                          4


5--Restriction-Ligation Reaction
ANNEAL ADAPTOR PAIRS
1. Calculate amounts of adaptor pairs needed (1 l of each pair per reaction multiplied
   by # samples)
2. Heat adaptor pairs separately in two 0.2 ml PCR tubes at 95C for 5 min in PE 2400
   thermal cycler [program “aflp-adaptors”]
3. Allow tubes to cool to room temperature (10 min)
4. Set aside

PREPARE ENZYME MASTER MIX
1. Calculate amounts by multiplying by # of samples; keep all reagents on ice; use
   within 1-2 hours of preparation
   a. 0.1 l 10X T4 DNA ligase buffer
   b. 0.1 l 0.5M NaCl
   c. 0.05 l 1mg/ml BSA
   d. 0.05 l (5 units) MseI
   e. 0.25 l (5 units) EcoRI
   f. 0.33 l (1 unit) T4 DNA ligase
   g. 0.22 l water
2. Combine above in 1.5 ml microfuge tube
3. Mix gently but thoroughly
4. Store on ice until ready

PREPARE RESTRICTION-LIGATION MASTER MIX
1. Calculate amounts by multiplying following by # of samples, plus 2 l extra; keep all
   reagents on ice; use immediately after preparation
   a. 1 l 10X T4 DNA ligase buffer
   b. 1 l 0.5M NaCl
   c. 0.5 l 1 mg/ml BSA
   d. 1 l MseI annealed adaptor pair
   e. 1 l EcoRI annealed adaptor pair
2. Combine above in 1.5 ml microfuge tube
3. Store on ice until ready

COMPLETE RESTRICTION-LIGATION
1. Over ice, for each sample combine in separate 0.2 ml PCR tube (total volume per
   sample 11.0 l):
   a. 1 l Enzyme Master Mix
   b. 4.5 l Restriction-Ligation Master Mix
   c. 5.5 l prepared DNA sample
2. Incubate at 37C for 2 hours in PE 2400 thermal cycler [program “aflp-digestion”]
3. Dilute each reaction with 189 l TE0.1 buffer; mix thoroughly
4. Store in refrigerator for up to one month, or freezer for longer
                                                                                       5


6--Pre-selective Amplification
PREPARE PRE-SELECTIVE AMPLIFICATION MASTER MIX
1. Calculate amounts by multiplying following by # of samples, plus 2 l extra; keep all
    reagents on ice; use immediately after preparation
    a. 7.5 l AFLP core mix
    b. 0.5 l AFLP pre-selective primer pairs

COMPLETE AMPLIFICATION
2. For each sample, combine in separate 0.2ml PCR tube (total volume per sample 10.0
   l):
   a. 8 l pre-selective master mix
   b. 2 l diluted restriction-ligation reaction
3. Amplify in PE 2400 thermal cycler [program “aflp-preselective”]
4. Store in refrigerator for up to one month, or freezer for longer

CONFIRM SUCCESSFUL AMPLIFICATION
1. Electrophorese 5 l of each pre-selective amp product in 1.3% agarose gel with EtBr,
   and 10 l of tracking dye/DNA ladder solution in leftmost well, for 15-20 min at ca.
   40 milli-Amps
2. Visualize under UV light
3. Successful amplification shows as a smear of product in 100-1500bp range


7--Selective Amplification
PREPARE TEMPLATE
1. For each pre-selective amplification product, combine in labeled 1.5 ml microfuge
    tube:
    a. 95 l TE0.1 buffer
    b. 5 l product
2. Mix thoroughly
3. Store in refrigerator for up to one month, or freezer for longer

PREPARE SELECTIVE AMPLIFICATION MASTER MIX
1. Calculate amounts by multiplying following by # of samples, plus 2 l extra; keep all
   reagents on ice; use immediately after preparation
2. For each prepared template, combine in 0.2ml PCR tube:
   a. 7.5 l AFLP core mix
   b. 0.5 l MseI (Primer-CXX)
   c. 0.5 l EcoRI (Dye-primer-AXX)

COMPLETE AMPLIFICATION
1. For each sample, combine in separate 0.2ml PCR tube (total volume per sample 10.0
   l):
   a. 8.5 l selective amplification master mix
   b. 1.5 l diluted pre-selective amplification product
                                                                                           6


2. Amplify in PE 2400 thermal cycler [program “aflp-selective”]
3. Store in refrigerator until ready to run on DNA sequencer


8--Sequence Analysis Preparation
In a labeled 1.5 ml microfuge tube for each sample, combine 0.5 l of each selective
amplification product (up to 3 different primer sets represented by 3 different colors).
Keep frozen until submitted to the sequencing technician.

Note: Sequencing technician performs the following to load the samples on the machine.

1. Prepare loading buffer by combining for each sample in a 1.5 l microfuge tube:
   a) 24 l formamide
   b) 1 l GeneScan-500 ROX standard
2. Pipette 25 l above loading buffer into each sequencer sample tube
3. Add selective amplification mixture submitted for analysis
4. Heat tubes to 95C for 3-5 min
5. Quick-chill on ice
6. Place sample tubes in machine sample tray and run
                                                                                         7


                                    APPENDIX I
                                  Supplies for AFLPs

Note: Selective primers (in italics) should not be purchased until initial screening with
the Selective Amp Start-up Module has been completed, and successful primer pairs with
the widest utility are identified.

                     Item                    Units     Unit Cost         Company
Ligation/Preselective Amp Module, 402004   100 rxns    $ 500       ABI
Selective Amp Start-up Module, 4303050     2000 rxns   $ 900       ABI
Amp Core Mix Module, 402005                500 rxns    $ 600       ABI
MseI-CAA Amp Primer, 402029                500 rxns    $ 140       ABI
MseI-CAC Amp Primer, 402028                500 rxns    $ 140       ABI
MseI-CAG Amp Primer, 402027                500 rxns    $ 140       ABI
MseI-CAT Amp Primer, 402026                500 rxns    $ 140       ABI
MseI-CTA Amp Primer, 402025                500 rxns    $ 140       ABI
MseI-CTC Amp Primer, 402024                500 rxns    $ 140       ABI
MseI-CTG Amp Primer, 402023                500 rxns    $ 140       ABI
MseI-CTT Amp Primer, 402022                500 rxns    $ 140       ABI
EcoR1-ACT FAM Amp Primer, 402037           500 rxns    $ 95        ABI
EcoR1-ACA FAM Amp Primer, 402030           500 rxns    $ 95        ABI
EcoR1-AAC NED Amp Primer, 4303054          500 rxns    $ 95        ABI
EcoR1-ACC NED Amp Primer, 4303056          500 rxns    $ 95        ABI
EcoR1-AGC NED Amp Primer, 4303058          500 rxns    $ 95        ABI
EcoR1-AAG JOE Amp Primer, 402034           500 rxns    $ 95        ABI
EcoR1-AGG JOE Amp Primer, 402035           500 rxns    $ 95        ABI
EcoR1-ACG JOE Amp Primer, 402036           500 rxns    $ 95        ABI
500 ROX standard, 401734                   800 rxns    $ 230       ABI

T4 DNA Ligase, M0202S                      20000 u     $    60     New England Biolabs
EcoR1 high (5X) conc, R0101T               10000 u     $    50     New England Biolabs
Mse1 high conc, R0525L                     1000 u      $   220     New England Biolabs
                                                                                           8


                                        APPENDIX I1
                                        AFLP Recipes

NaCl (0.5M)—makes 250 ml
7.31 g NaCl
250 ml autoclaved distilled water
Mix above well, store in refrigerator

1X TE0.1 buffer—makes 1l
2.42 g Tris
0.04 g EDTA
1 l autoclaved distilled water
Mix above well (may take 1 hr of intermittent stirring); aliquot into 250 ml bottles and
store in refrigerator

BSA (1mg/ml)—makes 10 ml
10 mg BSA
10 ml autoclaved distilled water
Mix above well; pipette 1000 l in each of 10 1.5 ml microfuge tubes, store in freezer