Bulk Segregant Mapping Protocols

ISOLATION OF INTACT GENOMIC DNA

Isolate genomic DNA from embryos using our genomic DNA isolation protocol.
 

BULK SEGREGANT ANALYSIS

All the mixes should be scaled up by the amount necessary for the number of samples to be analyzed.  Programs have been set up on the Beckman robot to aliquot the primers, set up the PCR reactions and to add loading buffer.

I. Preparation of Primers for PCR

For each PCR reaction, 0.2 µl of each primer (20 µM stock) will be used in a 10 µl reaction volume.  For easier distribution of the primers, they should first be diluted as follows:

To each well of a 96-deep well block add 460 µl of MilliQ water.  Then, to the appropriate well, add 20 µl of the forward and 20 µl of the reverse primer assigned to that position.  When the primers are then aliquoted, 5 µl of each primer mix will be added which is equivalent to 0.2 µl of the undiluted stock.

II. Primer Setup

The robot will be used to aliquot the primers from 96-well blocks.  The bulk segregant will be done on four sets of pools at a time to help in interpretation of the results (only one of the -/- pools would be expected to lack a band unless the mutations were linked).

Using the program BULKPRIM, 5 µl of the primers from column A1 to H1 will be aliquoted into columns A1-H4 of two plates (one for WT pools, the other for -/- pools); similarly, column A2 to H2 will be aliquoted into columns A5-H8 and column A3 to H3 will be aliquoted into columns A9-H12 of two plates.  Once the analysis has been done on these primer sets, it will be repeated for the primer sets in all wells of plates I and II.

III. Pooling of DNA

Combine 2 µl of the concentrated DNA from 24 WT embryos into a single 1.5µl tube (WT pool).
Combine 2 µl of the concentrated DNA from 24 mutant (-/-) embryos into a single 1.5µl tube (-/- pool).

Add 850ul of water to dilute each pool to a final volume of 900 µl.  This should provide more pooled DNA than actually required for the bulk segregant analysis.  Keep the diluted DNA at -20C for short term storage.  Remember to record which embryos were pooled to for the analysis (eg. -/- pool = A1-A12+B1-B12). < /FONT >

IV. Reaction Setup

To each of the wells, a pre-mix containing the DNA and rxn components will be added.
 
Component Per Reaction Single Pool Mix
10X PCR buffer A 
 		 1.0 µl 26.0 µl 
20 mM dNTPs  0.1 µl 2.6 µl
DNA pool 2.5 µl 65.0 µl 
dH2O 1.35 µl 35.1 µl 
Taq DNA polymerase (Fisher) 0.05 µl (0.25 U) 1.3 µl 
Total 10 µl  260 µl  (for 1 plate/1 pool) 
 
For each analysis, 8 pool mixes will be made as shown above (1WT + 1 -/- for 4 mutations = 8).  Depending on the number of plates per mutant to be analyzed, scale up the mixes accordingly. < /FONT >

Using the robot and program BULKPLS, 5 µl of the appropriate mix will be added to the aliquoted primers, as follows:
WT pool A to A1-H1, A5-H5, A9-H9
WT pool B to A2-H2, A6-H6, A10-H10
WT pool C to A3-H3, A7-H7, A11-H11
WT pool D to A4-H4, A8-H8, A12-H12

Similarly, for the -/- plate, add as follows:
-/- pool A to A1-H1, A5-H5, A9-H9
-/- pool B to A2-H2, A6-H6, A10-H10
-/- pool C to A3-H3, A7-H7, A11-H11
-/- pool D to A4-H4, A8-H8, A12-H12

The mixes should be made in individual 1.5 µl screw cap tubes and arranged on the robot as follows:
 
WT pool 1 WT pool 2 WT pool 3 WT pool 4    
-/- pool 1 -/- pool 2 -/- pool 3 -/- pool 4    
           
           
 

Seal plate and cycle using the CAS profile.  Once the cycle is complete, store at 4C or prepare for loading by adding loading buffer.  Use the robot and program BULKLB to add 5 µl of loading buffer per well to give a final volume of 15 µl.
 

V. Gel Analysis

The gel analysis of the PCR products will be performed using agarose gel electrophoresis and a mix of standard and MetaPhor agarose.

For each 100 ml of agarose, mix 1 g of MetaPhor + 1 g of standard agarose to give a 2% gel mix in 1X TBE.  Dissolve by heating to 50C but do not boil.  Extreme heat will damage the MetaPhor agarose and reduce resolving capacity.

Using the large electrophoresis chamber and gel tray with 96 well-spaced combs, load half (7 - 8 µl) of each sample, alternating between the WT and -/- plates (ie. load row A of the WT plate, then row A of the -/- plate, WT row B then -/- row B, etc.).  In this way, the WT and -/- pools will be loaded next to each other.

Run the gel at 150 to 200 V and allow bromphenol blue dye to migrate 3/4 of the way down the gel. 

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