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AGI BAC Filter Manual

The purpose of this manual is to explain what the filters are, clone designation procedure, what screening conditions to use, and clone analysis for the AGI Arabidopsis BAC library. Filter users are urged to send clone information for deposition into the BAC web depository.

I. What are the filters


You have received 3 Hybond N+ filters (approx 7.5 x 11 cm). Each filter has a label on the right side (usually a number and either 1-4, 5-8, or 9-12) and both the right upper and lower corners cut. The label will be on the DNA side of the filter. Each fi lter contains 384 3x3 grids, with filter 1-4 comprised of 384 well plates 1 to 4, filter 5-8 plates 5 to 8, and filter 9-12 plates 9 to 12. The grid pattern is described below for filter 1-4 and will increase the signal to noise since there should be two hybridization signals. Furthermore, the orientation of the signal will determine the 384 plate number. That is, a signal orientation of vertical on filter 1-4 would correspond to 384 well plate 2. Likewise, for filter 5-8 or 9-12 the 384 plate number woul d be 6 or 10, respectively.
1 2 3
     
4 - 4
     
3 2 1

II. Screening conditions


A. Prehyb filters at 65 C for 4 h with

0.5 M NaHPO4, pH 7.2

7% SDS

1% BSA - fraction V

1 mM EDTA

10 mg/ml sheared salmon sperm DNA

B. Exchange prehyb with fresh prehyb for 8 h at 65 C. These steps reduce background. For subsequent hybridizations you need only do step A.

C. Add probes (note: must use purified insert free of lacz sequence) and hyb for 18 to 36 h at 65 C.

D. Wash filters with 0.5x SSC and 0.1% SDS 3 times 20 mins at 65 C

E. Blot dry with paper towels, wrap in plastic wrap, and expose for 48 to 72 hours. NEVER let filter dry completely. This is important if you wish to reprobe.

F. Based on signal position, request clone from the ABRC at Ohio State University. For clones from 384 well plates, use TAMU <grid location>. For clones from 96 well plates use TAM <grid location>. We request that papers using TAMU BACs use the 384 well format in figures and text.

G. We advise that the filters NOT be stripped before reprobing.

H. This procedure, in our hands, gives an adequate backgound. However, users may wish to label BAC vector with 35S and include in the hyb solution. Or, addition of labeled plastid DNA and signal comparison with the plastid clone database should eliminate know plastid clones. By process of elimination, what is left should be your clone of interest.

III. Clone analysis


A. Streak clone on LB plus 12.5 ug/ml chloramphenicol plate and grow o/n at 37 C. Inoculate a single colony and grow 16 to 20 hr at 37 C.

B. Centrifuge entire o/n culture, pour out supernatant, resuspend pellet in remaining culture medium, recentifuge, remove supernatant, resuspend pellet in 0.2 ml.

50 mM glucose

10 mM EDTA

25 mM Tris, pH 8.0

5 mg/ml lysozyme

incubate on ice 5 min.

C. Add 0.4 ml 0.2 N NaOH, 1% SDS, mix gently, incubate on ice 5 min.

D. Add 0.3 ml KAc stock, mix gently, freeze -80 C 15 min.

E. Centrifuge 15 min in microfuge.

F. Remove 0.75 ml supernatant, transfer to new tube, add 0.45 ml isopropanol. Freeze -80 C 15 min. Centrifuge 5 min to pellet DNA.

G. Remove supernatant, rinse pellet with cold 70% EtOH. Centrifuge 2 mins.

H. Dry pellet on bench for 15 mins, add 40 ul TE (pH 8.0). Do not pipette. Let DNA dissolve o/n at 4 C.

I. Remove 10 ul with cut-off pipet tip (to prevent shearing), digest with 10 U NotI for 4 hrs at 37 C (total vol 40 ml). Run CHEF gel using standard conditions. We generally stain CHEF gels for 1 hr and destain for 2 hrs prior to photography

Conversion Protocol - 384 well plates to 96 well plates


In case you also have purchased the 96 well filters, here is a conversion protocol which simplifies the conversion between 384 well format and and 96 well format.

There are THREE simple steps

A. Determine which of the three filters your signal is on and then which 384 well plate.

B. The 384 grid location determines the 96 well plate identity.

C. Convert, if needed, the 384 grid location to 96 well format.

A. You should have three filters labeled 1-4, 5-8, or 9-12. Each filter has four 384 well plates gridded on it.
 
    Filter  
       
384 plate signal 1-4 5-8 9-12
diagonal left 1 5 9
vertical 2 6 10
diagonal right 3 7 11
horizontal 4 8 12

You'll need to know the 384 well plate number for the next step

B. The 384 well plate contains 4 96 well plates.

From the 384 well plate number and the grid location of the signal, determine the 96 well plate number from this table
    384 signal grid location    
         
  A1 to A12 A13 to A24 I1 to I12 I13 to I24
384 plate H1 to H12 H13 to H24 P1 to P12 P13 to P24
1 1 2 3 4
2 5 6 7 8
3 9 10 11 12
4 13 14 15 16
5 17 18 19 20
6 21 22 23 24
7 25 26 27 28
8 29 30 31 32
9 33 34 35 36
10 37 38 39 40
11 41 42 43 44
12 45 46 47 48

C. Convert the 384 grid location to 96 well grid, if needed.

This will need to be done if the letter is greater than H or the number is greater than 13 in the 384 grid location
  convert to       convert to  
I   A   13   1
J   B   14   2
K   C   15   3
L   D   16   4
M   E   17   5
N   F   18   6
O   G   19   7
P   H   20   8
        21   9
        22   10
        23   11
        24   12

EXAMPLES


384 well clone 11B3 becomes 96 well clone 41B3

384 well clone 6M21 bceomes 96 well clone 24E9

96 well to 384 well conversion protocol


The 384 well plates are gridded from A to P, 1 to 24, while 96 well plates are A to H and 1 to 12.
A1 ....... A12 A13 ...... A24
96 plate 1 96 plate 2
384 H1 H12 H13 H24
well I1 I12 I13 I24
grid 96 plate 3 96 plate 4
P1 ....... P12 P13 ...... P24

In other words, on 384 well plate 1, 96 well plate 1 is gridded between 384 wells A1 to A12, H1 to H12, 96 well plate 2 is gridded between 384 wells A13 to A24, H13 to H24, 96 well plate 3 is gridded between 384 wells I1 to I12, P1 to P12, 96 well plate 4 is gridded between 384 wells I13 to I24, P13 to P24

I. decide which 384 well plate contains your 96 well plate
96 well plate 384 well plate
1-4 1
5-8 2
9-12 3
13-16 4
17-20 5
21-24 6
25-28 7
29-32 8
33-36 9
37-40 10
42-44 11
45-48 12


II. Determine the 384 grid location from 96 well location

1. For 96 well plates 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, and 45: the 96 well location is identical with 384 well location, ie 9C22 becomes 3C22

2. For 96 well plates 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46: add 12 to the number, ie 96 well plate 2A4 becomes 1A16
1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18 19 20 21 22 23 24


3. For 96 well plates 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47: increase the letter designation by 8, ie 96 well plate11D9 becomes 3L9
A B C D E F G H
I J K L M N O P


4. For 96 well plates 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48: increase the letter designation by 8 AND increase the number by 12, ie 96 well 40F6 becomes 10N18
A B C D E F G H
I J K L M N O P
1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18 19 20 21 22 23 24
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A portion of AGI's material is based upon work supported by the National Science Foundation under Grant Number 102620.