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Project Documentation & Protocols: Maize Mapping Project: AFLP Protocols

AFLPs on the ABI 3100


The following protocols were developed for doing AFLP.s (or AFLP-MITEs) on an ABI3100 capillary sequencer. It is not recommended that AFLPs be done on an ABI 3700 sequencer. Many of the reagents for AFLPs are expensive and so every effort has been made to make the assay as cheap as possible while still maintaining high quality data. DNA quality and qualification is an important part of this procedure since the DNA used must be capable of being digested by restriction enzymes and being ligated efficiently to adapters. We have used the BRL plant DNA zol to prepare our DNA but phenol/CCl3 extracted CTAB DNA would probably work as well. All of our DNA is quantified using a fluorometer since in our hands has been more accurate than spectrophotometry. It is important that the DNA concentrations of all of the samples are more or less equal since this results in comparable AFLP profiles for all samples. The AFLP protocol is divided into: 1. Restriction/ligation; 2. Preselective amplification; 3. Selective amplification.

1. Restriction/Ligation Reaction

The restriction/ligation (R/L) reaction is done using the BRL core reagent kit but is modified to work with a fluorescent fragment detection system. Reaction volumes are minimized to conserve reagents and because the R/L and preselective amplification reactions generate more material than can be used. R/L reactions are generally done in a 96-well plate. A thermocycler with a heated lid works well since it minimizes evaporation of the small reaction volumes.

R/L Reaction Part 1 (restriction digestion)

# of Reactions

1 13.5 100 300
DNA @ 50 ng/ul 2 ul ---- ----* ----
5X Reaction Buffer 1 ul 13.5 ul 100 ul 300 ul
MseI/EcoRi mix 0.2 ul 2.7 ul 20 ul 60 ul
H2O 1.8 ul 24.3 ul 180 ul
540 ul
5 ul total 3 ul/rxt 3 ul/rxt 3 ul/rxt
volume

Let digest for 3 hours at 37 C. Heat kill the restriction enzyme by heating to 70 degrees C for 10 minutes. Cool and spin plate in centrifuge to bring down any condensation. Examine plate for excessive evaporation. Each well should have about 5 ul.

* It may be convenient to add the 100 ug of DNA to the plate and dry down. Dry at 37 C so as not to denature the DNA. In this event, the loss of water needs to be accounted for in the reaction volume so that you are adding 5 ul/reaction.

R/L Reaction, Part 2 (ligation reaction)

 

# of Reactions

1 13.5 100 300
BRL Ligation Bulk Mix 4.8 ul 64.8 ul 480 ul 1440 ul
T4DNA Ligase (1n/ul) 0.2 ul 2.7 ul 20 ul
60 ul
5 ul/rxt 5 ul/rxt 5 ul/rxt 5 ul/rxt
Let ligation reaction go at least 2 hours at 20 C or overnight. To the 10 ul R/L reaction add 30 ul of 0.1X T.E. to achieve a 1:4 dilution. Mix and seal plate. Store at .20 until ready to use.

Preselective Amplification (PSA) Reactions

The PSA described below is a 25ul reaction and will provide more than enough starting material when diluted 1:20. It is desirable to scale this back to a 10ul reaction if you do not want to visualize your PSA on a 1.5% agarose gel. If you do wish to run a gel, you will need to run at least 15ul to see anything, and even then, it will be very faint.

# of Reactions

1 13.5 100 300
DNA from R/L Reaction 2.5 ul ---- --- ---
BRL PSA primer mix 20 ul 270 ul 2000 ul 6000 ul
BRL PLATINUM TAQ
10X Buffer 2.5 ul 33.8 ul 250 ul 750 ul
BRL PLATINUM TAQ
(5u/ul) 0.1 ul 1.35 ul 10 ul 30 ul
MqClz (50mm) 1.25ul 16.9 ul 125 ul
375 ul
23.85ul/rxt 23.85 ul/rxt 23.85ul/rxt 23.85ul/rxt

Run the following PSA PCR program on the thermocycler.

94  3 minutes (To activate TAQ)
94  30 seconds**
56  60 seconds**
72  60 seconds**
72  5 minutes
4  Indefinitely
** 20 cycles each

Remove plate from the thermocycler and dilute 1:20. In general, 1-2 ul of this diluted PSA reaction is sufficient for a 10ul specific amplification.

 

Specific Amplification (SA)

The SA reaction is done in 96-well plates where 1-2 ul of PSA template has been added and dried down at 65 degrees C or less. The plates can then be stored at room temperature until needed. A Robins 96 hydra is used to dispense diluted PSA into the plates .

It works well to prepare a "generic" SA bulk mix that is "customized" by adding the EcoRi and MseI primers of interest. This allows a lot of reactions to be set up quickly. The generic bulk mix can be frozen and then be thawed when needed. The reaction described below are 10ul reactions but 5ul reactions would probably work as well.

# of Reactions

Bulk Mix 1 100 300
50mm MqCl(2) 0.36 ul 36 ul 108 ul
10X PCR Buffer 0.50 ul 50 ul 150 ul
H2O 6.14 ul 614 ul 1842 ul
7.0 ul/reaction

 

For:

# of Reactions

I Reaction 1 13.5 100 300
0.5 ul EcoRI dye labeled primer
(2pm/ul) 0.5 ul 6.8 ul 50 ul 150 ul
2.5 ul MseI primer
(6.7 ug/ul)/dNTP mix 2.5 ul 33.8 ul 250 ul 750 ul
0.05 ul BRL Platinum Taq (5u/lul) 0.05 ul 0.7 ul 5.0 ul 15.0 ul

 

MITE-AFLPs

An AFLP based assay has been developed for the detection of Miniature Inverted Repeat Transposable Elements (MITEs) in maize. MITEs tend to be located in the 31 end of genes and are relatively polymorphic across inbred lines. The MITE assay below was developed by the Wessler Laboratory for the "Heartbreaker" element family and would be adaptable to other MITE families (PNASV97, 18:10083-10089.) In the IBM population, over 200 polymorphic fragments were detected and mapped.

 

MITE Restriction/Ligation

# of Reactions

Prime Reaction 1 13.5 100 300
DNA @ 50 ug/ul*
10X One Phor All buffer 0.8 10.8 ul 80 ul 240
BSA @ 1 ug/ul 1.0 ul 13.5 ul 100 ul 300
MseI or BfaI @ 5 u/ul 0.08 ul 1.1 ul 8.0 ul 24.0 ul
100 mm DTT 0.4 ul 50.3 ul 40 ul 120 ul
H2O 3.72 50.3 ul 372 ul 1116 ul 6 ul/rxt
*

Incubate reaction at 37  for 3 hours. It may be convenient to work with DNA that has been dried down. If you do this, the water will need to be added to the reaction mix to compensate for the loss of volume.

# of Reactions

Ligation Reaction 1 13.5 100 300
10X OPA 0.2 ul 2.7 ul 20 ul 60 ul
BSA (1ug/ul) 1.0 ul 13.5 ul 100 ul 300 ul
10mm ATP 0.24 ul 3.25 ul 24 ul 72 ul
100 mm DTT 0.1 ul 1.35 ul 10 ul 30 ul
Ligase & weise unit/ul 0.2 ul 2.7 ul 20 ul 60 ul
MseI ADAPTERS @ 50pm/ul 0.2 ul 2.7 ul 20 ul 60 ul
H2O 0.06 ul
0.8 6 ul 18 ul
2 ul/rxt 2 ul/rxt 2 ul/rxt 2 ul/rxt
Ligate for 3 hrs to overnight at 37 C. Dilute the 10 ul R/L reaction 1:4 by adding 30 ul of 0.1X TE.
Preselective Amplification 1 13.5 100 300
Diluted R/L reaction 4 ul ---- ---- ----
MITE PS Hbr IntSE primer
(2pm/ul) + dNTPs 5 ul 67.5 500 1500
MITE PS MseI (or BfaI) primer 5 ul 67.5 500 1500
PLATINUM TAQ BUFFER (10X) 2.5 ul 33.8 250 750
BRL PLATINUM TAQ (5 u/ul) 0.10 ul 1.35 10 30
MaCl(2) (50mm) 0.75 ul 10.1 75 225
H2O 7.65 ul
103 765 2295
21 ul/rxt

 

MITE PRESELECTIVE PCR AMPLIFICATION

94 C 3 minutes

94 C 30 seconds**

59 C 30 seconds**

72 C 1 minute**

72 C 5 minutes

4 C Indefinitely

** 24 cycles each

The above reaction volumes are for a 25ul reaction and generate enough material so that 15ul can be run on a 1.5% agarose gel. Since 24 pcr cycles are done for the preselective amplification, 15ul should give a visible spread of DNA in the 100-1000 bp range. This is advisable since it provides a reliable check before proceeding to the selective amplification phase of the assay. Take the remaining 10ul and dilute with 190ul of 0.1XTE to give a 1:20 dilution. Use 1ul of this dilution for a 10ul selective amplification. In general, we use the Robbins Hydra 96 to dispense the 1ul into 96 well plates and dry the sample down in the oven. This makes for easy storage and setting up the selective amplifications can be done very quickly.

MITE SELECTIVE AMPLIFICATION

# of Reactions

 

1 13.5 100 300
MqCl2 (50mm) 0.36 ul 4.9 ul 36 ul 108ul
10X PLATINUM TAQ BUFFER 0.50 ul 6.8 ul 50 ul 150ul
H2O 6.1 ul 82.3 ul 610 ul
1830ul
7 ul/reaction
Customize the above reactions by adding:

 

# of Reactions

 

1 13.5 100 300
HbrInt5-F dy labeled primer
(2pm/ul) 0.5 ul 6.75 ul 50 ul 150 ul
MseI + N or BfaI + N primer
+ dNTPs 2.5 ul 33.8 ul 250 ul 750 ul
BRL PLATINUM TAQ 0.05 ul 0.7 ul 5 ul 15 ul

The above 10ul reactions are dispensed into a 96-well plate in which 1 ul of the diluted preselective amplification has been dried down. It is likely that 5ul reactions would work as well and would conserve reagents.

 

MITE Selective Amplification

94 C 3 minutes . to activate TAQ
94 C 30 seconds **
70 C 30 seconds **
72 C 60 seconds **
72 C 45 minutes
4 C Indefinitely
** Touchdown at 1 C/cycle to 61 C
29 total cycles at 61 C

 

 


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