PIF 3 and TPI qPCR on D. innubila injected with dilutions of DiNV from day 0, 1, 3, and 5 set 1 and 2

Sample information can be found here, and injection information is here

Samples were thawed on ice and vortexed and spun down. Samples were placed in order for qPCR in 2 96 well plates and diluted 1:10 needed for qPCR: 27ul of DNA hydration solution and 3ul of DNA. These were then frozen until use for qPCR. Plates were sealed with 8 strip caps.

Plate layouts:
Plate 1

  1 2 3 4 5 6 7 8 9 10 11 12
A 29 21 11 1 45 37 63 53 82 72 99 91
B 116 107 135 126 153 144 169 161 189 178 209 199
C 31 23 13 3 47 39 65 55 84 74 101 93
D 118 109 137 128 155 146 171 163 191 180 210 201
E 33 25 15 4 49 41 67 57 86 76 103 95
F 120 111 139 130 157 148 173 165 193 182 211 203
G 35 27 17 5 51 43 69 59 88 78 105 97
H 122 113 141 132 159 150 175 167 195 184 212 205

Plate 2

  1 2 3 4 5 6 7 8 9 10 11 12
A 30 22 12 2 46 38 64 54 83 73 100 92
B 117 108 136 127 154 145 170 162 190 179 213 200
C 32 24 14 6 48 40 66 56 85 75 102 94
D 119 110 138 129 156 147 172 164 192 181 214 202
E 34 26 16 7 50 42 68 58 87 77 104 96
F 121 112 140 131 158 149 174 166 194 183 215 204
G 36 28 18 8 52 44 70 60 89 79 106 98
H 123 114 142 133 160 151 176 168 196 185 216 206

The plan is to do every sample only once (no tech reps in qPCR) but there will be 8 bio reps per treatment

qPCR of Plate 1 with PIF3 and TPI

  • Thawed plate 1 on ice, pipette mixed each well, then spun down
  • Prepared mixes for qPCR on ice, everything was thawed on ice, vortexed and spun down before use:
reagent PIF 3 TPI
SSo 520ul 520ul
F primer 52ul 52ul
R primer 52ul 52ul
molec grade water 312ul 312ul
  • 9ul of PIF 3 mix was added to each well in a qPCR plate
  • Added 1ul of diluted sample to each well of the plate using a multichannel: using Plate 1
  • Pipette mixed each well with the multichannel
  • Sealed the plate
  • Spun down the plate at 4,000rpm for 5 min
  • Placed the plate in the qPCR machine with the KMM-P47 program
  • This ran for ~1.5 hours, in the last half hour I prepared the TPI plate in the same way as above
  • I also used only plate 1 DNA
  • Ran the TPI plate on the same KMM-P47 program

qPCR data for plate 1 can be found here

qPCR of Plate 2 with PIF3 and TPI was done on 20240225

Thawed plate 2 on ice, pipette mixed each well, then spun down

  • Prepared mixes for qPCR on ice, everything was thawed on ice, vortexed and spun down before use:
reagent PIF 3 TPI
SSo 520ul 520ul
F primer 52ul 52ul
R primer 52ul 52ul
molec grade water 312ul 312ul
  • 9ul of PIF 3 mix was added to each well in a qPCR plate
  • Added 1ul of diluted sample to each well of the plate using a multichannel: using Plate 2
  • Pipette mixed each well with the multichannel
  • Sealed the plate
  • Spun down the plate at 4,000rpm for 5 min
  • Placed the plate in the qPCR machine with the KMM-P47 program
  • This ran for ~1.5 hours, in the last half hour I prepared the TPI plate in the same way as above
  • I also used only plate 2 DNA
  • Ran the TPI plate on the same KMM-P47 program

qPCR data for plate 2 can be found here

Analysis with all data is here