 |
|
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 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.
R/L Reaction, Part 2 (ligation reaction)
# of Reactions 1 13.5 100 300 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 Run the following PSA PCR program on the thermocycler. 94 3 minutes (To activate TAQ) 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
For: # of Reactions I Reaction 1 13.5 100 300
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 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
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 1 13.5 100 300
# of Reactions 1 13.5 100 300 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
Return to Documentation Index | Return to Maize Mapping Project Index | Return to Homepage |
|
If you have some comments about this page, or about the site in general, fill out our easy to use feedback form without leaving this page! |
Be sure to cite us! |
|