Daniel:Notebook/ComboLock/2016-11-16

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BSA Beads Test (Started Yesterday)[edit]

Back to Calendar

Protocol[edit]

  1. Rolling Circle Amplification
    1. Prepare 10.2X Master mix according to table below
    2. Add sample to new reaction tubes according to following table
    3. Reagent uL Added Master Mix (10.2X)
      Template 5 NA
      RCA Primer (100 uM) 2.5 25.5
      dNTP (1 mM) 0.8 8.16
      10X Buffer 2 20.4
      Phi29 1 10.2
      nfH2O 8.7 88.74
      Total 20 153
    4. Incubate at 37C for 3 hours
    5. Incubate at 65C for 10 minutes;Hold at 10C until next step
  2. qPCR
    1. Make master mix according to following recipe
      1. 422.1 uL nfH2O
      2. 502.5 uL SYBR Fast
      3. 20.1 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section
    4. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  3. TBE Gel
    1. Mix 200 uL TBE, 50 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]

qPCR Sample Table[edit]

Sample RCA-Primer Condition Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
Sample 1AC Chris 2 ug BSA-biotin A1 20 2 25 1 1 21 50
Sample 1BC Chris 2 ug BSA-biotin A2 20 2 25 1 1 21 50
Sample 2AC Chris No BSA-biotin A3 21 2 25 1 1 21 50
Sample 2BC Chris No BSA-biotin A4 21 2 25 1 1 21 50
Sample 3AC Chris No Antibody A5 22 2 25 1 1 21 50
Sample 3BC Chris No Antibody A6 22 2 25 1 1 21 50
Sample 4AC Chris 20 ng BSA-biotin A7 23 2 25 1 1 21 50
Sample 4BC Chris 20 ng BSA-biotin A8 23 2 25 1 1 21 50
Sample 5AC Chris 200 pg BSA-biotin D1 24 2 25 1 1 21 50
Sample 5BC Chris 200 pg BSA-biotin D2 24 2 25 1 1 21 50
Sample 1AD Dan 2 ug BSA-biotin D3 20 2 25 1 1 21 50
Sample 1BD Dan 2 ug BSA-biotin D4 20 2 25 1 1 21 50
Sample 2AD Dan No BSA-biotin D5 21 2 25 1 1 21 50
Sample 2BD Dan No BSA-biotin D6 21 2 25 1 1 21 50
Sample 3AD Dan No Antibody D7 22 2 25 1 1 21 50
Sample 3BD Dan No Antibody D8 22 2 25 1 1 21 50
Sample 4AD Dan 20 ng BSA-biotin H1 23 2 25 1 1 21 50
Sample 4BD Dan 20 ng BSA-biotin H2 23 2 25 1 1 21 50
Sample 5AD Dan 200 pg BSA-biotin H3 24 2 25 1 1 21 50
NTC     H4 25 0 25 1 1 23 50

Library Prep (Started Yesterday; Exp. Start 11/7/16)[edit]

  1. Ethanol Precipitation
    1. Centrifuge for 25 minutes at 12000 rpm at 4C
    2. Remove supernatant and add 750 uL 70% EtOH
    3. Centrifuge for 12 minutes at 12000 rpm at 4C
    4. Remove supernatant and spin in vacuum centrifuge for 10 minutes
    5. Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A and B by reusing the water
Sample ng/uL ug in 30 uL
Cells RCA Product (2AB,2BB) 47.8 1.4

DEJ-CL-CellC01-20161116-Ind25