Tuesday 9/22/15

Lab Work

Set up a test PCR to determine the optimum # of cycles. You want to identify the minimum number of cycles required for a visible product at 166 bp. I chose 4 of the samples and tested them at 12, 17, 22, & 27 cycles as recommend in the protocol.

  • Made 100 uM stock of new ILL-Lib2 adaptor
  • Made 10 uM stocks of ILL-Lib1 and ILL-Lib2 primers
    • 1.5 stock + 13.5 uL NFW
  • Made 1 uM stocks of HT1 and BC1
    • 1uL stock + 99 NFW
1x
17x
NFW
4.6 uL
78.2
10 mM (each) dNTPS
.4
6.8
10 uM ILL-Lib1
.4
6.8
10 uM ILL-Lib2
.4
6.8
1 uM ILL-HT1
1
17
1 uM ILL-BC1
1
17
5X Q5 buffer
4
68
Q5 Taq polymerase
.2
3.4
Added 12 uL master mix to 8 uL ligation.
  1. SS2_3_12x
  2. HC1_1_12x
  3. NF1_1_12x
  4. NF1_2_12x
  5. SS2_3_17x
  6. HC1_1_17x
  7. NF1_1_17x
  8. NF1_2_17x
  9. SS2_3_22x
  10. HC1_1_22x
  11. NF1_1_22x
  12. NF1_2_22x
  13. SS2_3_27x
  14. HC1_1_27x
  15. NF1_1_27x
  16. NF1_2_27x

17 and 18 are 2 of the ligation reactions for comparison.

  • 17: SS_2_3
  • 18: NF_1_1
Programed PCR in thermocyclers 4,5,6. Called 2b12, 2b17, 2b22, and 2b27.
  •  (98°C 5 sec, 60°C 20 sec, 72°C 10 sec) X N cycles

9_22_15

It looks like 22 cycles is best, and worked on all samples so will be using that in the preparatory scale.

Ethanol precipitation

  • Set up an ethanol precipitation of all the broodstock samples I’ve extracted so far, as well as repeats of of the 4 samples used in the test scale PCR:
    • Population Sample Date extracted ng/uL Volume for 1 ug Volume sodium acetate Vol ethanol
      Oyster Bay BS_2_3 19.8 50.50505051 5.05 113.64
      Hood Canal BS_1_1 12.3 81.30081301 8.13 182.93
      Fidalgo BS_1_1 9.13 109.5290252 10.95 246.44
      Fidalgo BS_1_2 17.4 57.47126437 5.75 129.31
      Oyster Bay BS_2_6 23.4 42.73504274 4.27 96.15
      Oyster Bay BS_2_7 16.4 60.97560976 6.10 137.20
      Oyster Bay BS_2_8 16.8 59.52380952 5.95 133.93
      Hood Canal BS_1_5 12.1 82.6446281 8.26 185.95
      Hood Canal BS_1_6 23.5 42.55319149 4.26 95.74
      Hood Canal BS_1_7 14 71.42857143 7.14 160.71
      Hood Canal BS_1_8 22.8 43.85964912 4.39 98.68
      Hood Canal BS_1_9 13.9 71.94244604 7.19 161.87
      Hood Canal BS_1_10 27.7 36.10108303 3.61 81.23
      Fidalgo BS_1_4 8.9 112.3595506 11.24 252.81
      Fidalgo BS_1_5 18.1 55.24861878 5.52 124.31
      Fidalgo BS_1_6 16.1 62.11180124 6.21 139.75
      Fidalgo BS_1_7 35.8 27.93296089 2.79 62.85
      Fidalgo BS_1_8 22.7 44.05286344 4.41 99.12

      Left in ethanol and sodium acetate in -20C overnight.

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One thought on “Tuesday 9/22/15

  1. Pingback: Wednesday 9/23/15 | Getting My Genes Wet

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