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Project Documentation & Protocols: Maize Mapping Project: SSR Protocols

DNA

DNA for SSR PCR may be extracted using one of the many isolation methods.

CTAB extraction based on the method of Saghai-Maroof et al. will supply more than enough very clean DNA. The microextraction method is an excellent, very quick method, especially if there are many individuals (100+) in the population. Though the CTAB method is much more time consuming, CTAB extracted DNA yield is higher than microextracted DNA and may remain intact, undegraded, longer, although I have gone back to a plate of diluted DNA more than a year after initial prep and it works.

The working concentration of the DNA should be 10 ng/µl in sterile water. Hydrolytic activity will cause the DNA to degrade at this low concentration. The best practice is to maintain a concentrated stock (at least 200 ng/µl) in TE and then dilute in sterile water an amount (10 ng/µl) that will be used rather quickly. Repeated freezing and thawing of the diluted DNA is also detrimental to quality. Once the dilutions have been made, store the concentrated DNA at ~20° C and store the working stock plate at +4° C.

We use a 96-well polypropylene (800µl capacity/well) with a tight fitting mat-lid (Matrix Technologies) for the DNA stock plate. If there are 25-30 primer sets to map, 2 µg of DNA/well is diluted to the working concentration for a total of 200 ml of diluted DNA. At 5 ml diluted DNA per reaction (50 ng), this is enough for 35+ primer sets, with allowances for pipetting errors or evaporation. To facilitate aliquotting of the DNA and the bulk mix of PCR reagents as well as the loading of the gels, the entire system is set up in the 96-well format and a 12-tip multichannel pipettor is used. For lab note purposes, a map (or TITER PLATE SET UP ) showing what each well of the microtiter plate contains is helpful for set-up.

PRIMER SETS

Some of SSR primer sets may be purchased from Invitrogen, with trade names Mappairs for Satellites, but we recommend that you get the primers synthesized by the supplier that is your own personal favorite. MaizeGDB has a listing of all public SSR primer sets for maize. There are many companies that can synthesize oligonucleotides less expensively. If the primer set nucleotide sequence is published, you may request any one of these other companies to produce the oligonucleotides. They will synthesize many more micrograms of primer. This is very cost effective if the primer set is polymorphic for more than one population, and will provide more than an ample amount of primer for screening and mapping multiple populations. Generally, these other companies send lyophilized pellets of oligonucleotides. An easy way to deal with these is to dilute them to 1µg/µl in TE and then dilute a working stock (500 µl or less) to 50 ng/µl in sterile water.

PCR

Polymerase chain reaction (PCR) conditions and cycling profile are based on the protocol established by E. Lynn Senior but include slight modifications, mainly to accommodate the specific Taq polymerase used for the experiment, and are as follows (final concentrations):

PCR Buffer 1X, final reaction concentrations

10 mM Tris-HCl pH 8.3

50 mM KCl

2 mM MgCl2

0.001% gelatin

0.2 mM each dNTP

0.02 unit/µl Taq, inactive until initial denaturation or set up reactions on ice

10-30 ng genomic dna

All thermocycling is performed in a 96-well thin-walled microtiter style plate with an oil overlay (13µl makes a drop that will come off the pipet tip without having to touch the components in the well) in an Amplitron II (Barnstead-Thermolyne) or (MJ Research) Tetrad. The cycling profile includes a preliminary eight to ten minute dwell at 95° C to activate the AmpliTaq Gold. NOTE, the BRL Platinum Taq does not require this.

95° C 1 minute

65° C 1 minute

72° C 1 minute 30 seconds

for one cycle and then a one-degree decrement for the annealing temperature, each repeated once, until the annealing temperature is 55° C. The regime is then,

95° C 1 minute

55° C 1 minute

72° C 1 minute 30 seconds

repeated for a total of 30 cycles.

SCREENING

Like RFLP, SSR primer sets must be screened against the parents in the population of interest. It is also imperative to include the F1 in the screening process as well, to confirm the presence of parental bands in the F1. There are a frustrating percentage of primer sets that amplify different band sizes between the parents that are not represented in the F1. In some cases, there will be a third band in the F1 (also in the F2's), these are useful as long as the parental bands are represented within the three bands. During screening, when using the 96-well format, it is most economical to combine the 1 µl forward and 1µl reverse primer and pipette the 2 µl mixture into the well. Add the 5 µl (50 ng) DNA next, and finally, make a bulk mix of the remaining components and (multichannel) pipet these into the wells.

The 3.5% SFR gels should be loaded and run with constant voltage of 115-130V and checked every 90 minutes. It is most helpful to take a picture of the gel at each time point to determine polymorphism and the best run length. Most maize SSR polymorphism band sizes range between 90 basepairs (bp) and 180 bp and are well resolved within 2-3 hours at 115 V. Be patient during the screening process, most of these amplified bands are only 5-10 bp different and if they are higher molecular weight, they will not immediately separate in the 4% SFR. Again, keep track of the times necessary to detect polymorphism for future use in mapping. A few primer sets generate bands that are 200-300 bp in size. They will separate, eventually, in a 4% gel, but if they are to be used for mapping, pour a 3% gel. A 5% gel will be good for bands less than 100 bp, but not really necessary.

GELS

SFR agarose (Amresco) is used to resolve the amplified bands. (Metaphor agarose is an equivalent quality brand from the company FMC.) A 4% SFR (Super Fine Resolution) gel resolves 50-500 bp and is approximately equivalent to a 6% acrylamide gel. The first time a gel is made, follow the manufacturer's recommendations for slowly adding the powder agarose to the fast swirling 1X TBE and heating very slowly. Make a mark on the side of the flask to indicate the level of the agarose, stir bar plus the 1X TBE. The first time this agarose is melted, it tends to trap much air and overboil quickly. Be very careful!! After completely melting, put the agarose back on the stir plate and stir for 10 minutes. Add ethidium bromide to 2 µg/ml (for the first time ONLY). Pour the gels when the agarose is cool enough that you can comfortably hold the bottom of the flask to your palm. This agarose may be reused numerous times and prior to repouring, will need only a little more ethidium (5-10 µl) after each remelting. After remelting, always check that the level makes the mark on the side of the flask, if not, add 1X TBE. We have used many gels over 40 times each. The only reason to stop reusing the gel is when an undesirable amount of background fluorescence builds up.

In the 12-channel pipettor set-up, we have found the BRL Sunrise 96 gel boxes with the 26-well combs to be very useful. The 12-channel pipettor is spaced such that it loads every other well of the 26-well comb. Therefore, the titer plates must be organized accordingly. The 'A' row of the titer plate would be loaded into lanes 2, 4, 6, and 24 and the 'B' row would be lanes 3, 5, 7, and 25. Confusing as it seems on paper, this will make much more sense once you've set up a plate and worked with it. From the titer plate to the gel, each pair of rows of the titer plate (A & B, C & D, etc.) correspond to one tier of the gel, loaded so that there is an empty well at the beginning and the end of the tier. C & D rows are tier 2 and so on, to rows G & H. The empty well may be used for a molecular weight marker. If multiple plates are required to represent the population, it is a good idea for tracking purposes, to load a molecular weight marker on different tiers of the gel. For example, load a marker on the first tier to indicate the first titer plate, load on the second tier for the second plate of the population. The marker should have bands every 100 bp, starting at 50 or 100 bp. Because of the high percentage agarose, bands much over 1000 bp will not separate.

MAPPING

Once polymorphism has been identified, the population should be set up in the microtiter stock plates. The bulk mix can now include the primer set. For larger (more than one microtiter plate) populations, the PCR reagents can be mixed in bulk in a reagent boat, a V-bottom plastic tray the width of the multichannel pipet. This is where the multichannel pipettor is especially appreciated. 10 µl of bulk mix is pipetted into the PCR titer plate (no need to change tips) and 5 µl of DNA is added (change tips every time!). A 13 µl mineral oil drop is added (no tip change if you don't touch anything) to prevent the reaction mixture from boiling away. All of this is done with the multichannel. Gel properties are as mentioned above, but only need to be run until polymorphism is detectable and individuals can be scored

TITER PLATE SET UP for 12-tip multichannel pipette

a1

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h1

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10X TBE
108 g Tris-base
55 g boric acid
9.3 g disodium EDTA
pH should be around 8.3 without adjustment
bring volume up to 1 L


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