AGI Protocols

How to identify an address:

The membrane is divided into 6 fields (diagram provided). Each field contains 384 squares. The 384 squares represent the row and column identification of the BAC. Within each square there are 16 positions where 8 clones are spotted in duplicate (diagram). The pattern of the spotted clones will generate the plate address of the BAC. To identify your clone, please follow the directions below.

The most complicated part about identifying a clone address is that consecutive plates are not spotted into each field. The 384 well plates are spotted onto the membrane with plates 1-6 spotted into fields 1-6 respectively (duplication pattern 1, see diagram). Since there is a total of 6 fields on the membrane, the cycle will continue with the next six consecutive plates (plates 7 through 12) again being spotted into fields 1 through 6 respectively, but in a different duplication pattern (duplication pattern 2, see diagram). This gridding cycle will continue until all the plates have been spotted.

1. The library name and filter number is used to orient the membrane. Place the membrane with the label facing up and on the right-hand side as shown in the diagram. (The colonies are on the same side of the filter as the label)
2. Identify the field number of the hybridizing colonies. The spacing of colonies is slightly wider between the fields.
3. Identify the well location (I have included a grid to help locate well positions) and identify the well position (e.g. L18)
4. Identify the plate number. This is accomplished by determining the orientation of the duplicate spots (duplication pattern in the diagram) and referring to the table inside each field in the figure.
5. Libraries which have more than one filter (48 plates) will also need to decode the plate number based on the filter. Plates 1-48 are spotted on filterA, 49-96 on filterB, etc. Identify the plate number and well location as described above and record the filter letter. Go to the conversion table and read down the column corresponding to the filter letter and read across the plate number identified from the filter. The intersection is the actual plate number of the clone.

Example: If you have horizontal spots, they could either be duplication position 4 or 8 (from the duplication pattern). They are distinguished by the closeness of the spots and position in the pattern. Assume it is position 4 in field 3. Read down the table in field 3 of the diagram to pos4 and read the plate number as 21. If you are reading filterB, identify the library plate number from row 21 and the column labeled FilterB of the library filter plate decoder. The library plate number is 69. Once the plate number is determined, identify the well location either by using the supplied grid or counting the rows and columns.

Click here to see the 4 X 4 Six Fields Example

Click here to see the 4 X 4 Four Fields Example

Click here to see the Library Plate Decoder (Excel Spreadsheet)

Click here to see the Library Plate Grid

Hybridization Protocol

Solutions:

1. 1M Sodium Phosphate
2. 134 g Na2HPO4.7H2O
3. pH to 7.2 with 3.5 - 4 ml H3PO4
4. adjust to 1L

Hybridization Solution:

1. 160 ml 1M sodium phosphate
2. 112 ml 20% SDS
3. 0.6 ml 0.5M EDTA
4. add water to 320 ml

Prehybridization:

Add 30 ml of hybridization buffer and 300 ul of 10 mg/ml denatured salmon sperm DNA to a 300 ml hybridization bottle. Incubate filters overnight at 65 C.

Hybridization:

Discard prehybridization solution and add fresh hybridization solution containing denatured probe. Incubate overnight at 65 C.

Washing:

Wash twice with: 1X SSC, 0.1% SDS 65 C for 20 min. If the signal is very strong, proceed with the next washes.

Wash twice with: 0.5X SSC, 0.1% SDS 65 C for 20 min.

After washing, wrap the filter in saran wrap and expose to film. Make alignment spots through the film and into the filter to assist in aligning the film after developing.

Membrane Stripping Protocol

1. Wash for 10 min at room temperatue with shaking in 100 mM NaOH, 10 mM EDTA, 0.1% SDS
2. Repeat the washing from step one
3. Quickly rinse the membarane in deionezed water (30 sec to 1 min)
4. Wash membrane in 5X SSPE for 10 min
5. Repeat the washing from step 4