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
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1x
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17x
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NFW
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4.6 uL
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78.2
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10 mM (each) dNTPS
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.4
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6.8
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10 uM ILL-Lib1
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.4
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6.8
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10 uM ILL-Lib2
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.4
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6.8
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1 uM ILL-HT1
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1
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17
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1 uM ILL-BC1
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1
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17
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5X Q5 buffer
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4
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68
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Q5 Taq polymerase
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.2
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3.4
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Added 12 uL master mix to 8 uL ligation.
- SS2_3_12x
- HC1_1_12x
- NF1_1_12x
- NF1_2_12x
- SS2_3_17x
- HC1_1_17x
- NF1_1_17x
- NF1_2_17x
- SS2_3_22x
- HC1_1_22x
- NF1_1_22x
- NF1_2_22x
- SS2_3_27x
- HC1_1_27x
- NF1_1_27x
- 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
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:
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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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