Daniel:Notebook/ComboLock/2016-11-4

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Antibody Binding Test (Started Yesterday)[edit]

Back to Calendar

Protocol[edit]

  1. qPCR
    1. Make master mix according to following recipe
      1. 233.1 uL nfH2O
      2. 277.5 uL SYBR Fast
      3. 11.1 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 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
  2. TBE Gel
    1. Mix 120 uL TBE, 30 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

qPCR Sample Table[edit]

Sample 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 1A 2 ug BSA A1 20 2 25 1 1 21 50
Sample 1B 2 ug BSA A2 20 2 25 1 1 21 50
Sample 2A No Template A3 21 2 25 1 1 21 50
Sample 2B No Template A4 21 2 25 1 1 21 50
Sample 3A No Antibody A5 22 2 25 1 1 21 50
Sample 3B No Antibody A6 22 2 25 1 1 21 50
Sample 4A 20 ng BSA A7 23 2 25 1 1 21 50
Sample 4B 20 ng BSA A8 23 2 25 1 1 21 50
Sample 5A 200 pg BSA H1 24 2 25 1 1 21 50
Sample 5B 200 pg BSA H2 24 2 25 1 1 21 50
NTC qPCR NTC H3 25 0 25 1 1 23 50

Results[edit]


Testing[edit]

Doing some QC control on the antibody experiment to figure out what went wrong. The first thing to test is the BSA Ab-oligo complexes.

  1. qPCR
    1. Make master mixes according to following recipes
    2. Master Mix No. Samples Primers uL Reverse Primer (10 uM) uL Forward Primer (10 uM) uL Kapa SYBR Fast uL nfH2O
      PA 10 p24/p4RC 10.1 10.1 252.5 212.1
      PB 2 p6/p12RC 2.1 2.1 52.5 44.1
      Nonsense A 2 p24/p12rc 2.1 2.1 52.5 44.1
      Nonsense B 2 p6/p4RC 2.1 2.1 52.5 44.1
    3. Add 47 uL master mix to each well
    4. Add 2 uL sample according to table in following section
    5. 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
  2. TBE Gel
    1. Mix 160 uL TBE, 40 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 Primers Primer Target Time Point Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
BSA-Ab p24/p4RC PA NA A1 2 25 1 1 21 50
BSA-Ab p6/p12RC PB NA A2 2 25 1 1 21 50
BSA-Ab p24/p12rc Nonsense NA A3 2 25 1 1 21 50
BSA-Ab p6/p4RC Nonsense NA A4 2 25 1 1 21 50
Sample1A p24/p4RC PA T1 A5 2 25 1 1 21 50
Sample1B p24/p4RC PA T1 A6 2 25 1 1 21 50
Sample2A p24/p4RC PA T2 A7 2 25 1 1 21 50
Sample2B p24/p4RC PA T2 A8 2 25 1 1 21 50
Sample3A p24/p4RC PA T3 H1 2 25 1 1 21 50
Sample3B p24/p4RC PA T3 H2 2 25 1 1 21 50
Sample4A p24/p4RC PA T4 H3 2 25 1 1 21 50
Sample4B p24/p4RC PA T4 H4 2 25 1 1 21 50
NTC p24/p4RC PA NA H5 0 25 1 1 23 50
NTC p6/p12RC PB NA H6 0 25 1 1 23 50
NTC p24/p12rc Nonsense NA H7 0 25 1 1 23 50
NTC p6/p4RC Nonsense NA H8 0 25 1 1 23 50