Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 1

I plan to pick 94 colonies from plate 1 using a autoclaved pipette tip for each one, first dap that tip in a new gridded LB plate, then dunk that tip in a 96 well plate with water to create a bacteria solution to do PCR on.

  • First, made 4 new LB plates with chloramphenicol and kanamycin
    • Made a solution of 45ul 100% ethanol and 45ul of stock chlor
    • Spread 20ul of the chlor solution on 4 new LB plates
    • Then spread 20ul of stock kan on the same plates
    • Let the plates dry a little in the bacteria hood
    • These plates had grids on them with numbers to keep track of the colonies (see below)
  • Prepared a 96 well plate with strip caps with 5ul of molecular grade water in each well
  • For each colony in plate 1 from the fridge:
    • I picked it up with an autoclaved p2 tip
    • I dabbed it lightly on the appropriate square in the gridded plate
    • I swirled it for a few seconds in the appropriate well in the 96 well plate
  • This took about and hour to 2 hours

The gridded plates were numbered like this:

Plate 1A:

1 2 3 4 5
6 7 8 9 10
11 12 13 14 15
16 17 18 19 20
21 22 23 24 25

Plate 1B:

26 27 28 29 30
31 32 33 34 35
36 37 38 39 40
41 42 43 44 45
46 47 48 49 50

Plate 1C:

51 52 53 54 55
56 57 58 59 60
61 62 63 64 65
66 67 68 69 70
71 72 73 74 75

Plate 1D:

76 77 78 79 80
81 82 83 84 85
86 87 88 89 90
91 92 93 94  
         

And the 96 well plate:

  1 2 3 4 5 6 7 8 9 10 11 12
A 1 9 17 25 33 41 49 57 66 74 82 90
B 2 10 18 26 34 42 50 58 67 75 83 91
C 3 11 19 27 35 43 51 59 68 76 84 92
D 4 12 20 28 36 44 52 60 69 77 85 93
E 5 13 21 29 37 45 53 61 70 78 86 94
F 6 14 22 NA 38 46 54 62 71 79 87 X
G 7 15 23 31 39 47 55 63 72 80 88 X
H 8 16 24 32 40 48 56 65 73 81 89 X

For some reason sample 64 got lost and did not end up in this plate

PCRs

  • Then, the solution in the 96 well plate was used for colony PCR
  • The general PCR protocol was used, however 1ul of the bacteria-water solution was used instead of DNA
  • I used lef 4 as the primer to amplify DiNV DNA because it had not shown any off-target amplification before
  • 16S is a control for bacteria presence in the sample
  • PCR mixes:
reagent lef 4 16S
GoTaq 500ul 500ul
F primer 25ul 25ul
R primer 25ul 25ul
molec grade water 350ul 350ul

Reactions were run for 30 cycles. Primer information can be found here. The gels from this plate can be found here. No colony was positive for DiNV.