Daniel:Notebook/ComboLock/2017-1-28

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RCA Timing Test (v3 Oligos)[edit]

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Protocol[edit]

  1. qPCR
    1. Make qPCR master mix according to following recipe
      1. 361.2 uL nfH2O
      2. 17.2 uL AmpF6.4
      3. 430 uL Kapa SYBR Fast
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL AmpR indexed 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 280 uL TBE, 70 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 Table[edit]

Sample Sample Condition AmpR Index Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
Sample 1A Sample PreRCA 10 A1 2 25 1 1 21 50
Sample 1B Sample PreRCA 10 A2 2 25 1 1 21 50
Sample 2A No Template PreRCA 11 A3 2 25 1 1 21 50
Sample 2B No Template PreRCA 11 A4 2 25 1 1 21 50
Sample 3A No C Probe PreRCA 12 A5 2 25 1 1 21 50
Sample 3B No C Probe PreRCA 12 A6 2 25 1 1 21 50
Sample 4A No PO4 PreRCA 13 A7 2 25 1 1 21 50
Sample 4B No PO4 PreRCA 13 A8 2 25 1 1 21 50
Sample 1AX Sample 3 hours 14 H1 2 25 1 1 21 50
Sample 1BX Sample 3 hours 14 H2 2 25 1 1 21 50
Sample 2AX No Template 3 hours 15 H3 2 25 1 1 21 50
Sample 2BX No Template 3 hours 15 H4 2 25 1 1 21 50
Sample 1AY Sample 6 hours 16 H5 2 25 1 1 21 50
Sample 1BY Sample 6 hours 16 H6 2 25 1 1 21 50
Sample 3AY No Template 6 hours 17 H7 2 25 1 1 21 50
Sample 3BY No Template 6 hours 17 H8 2 25 1 1 21 50
Sample 1AZ Sample 12 hours 18 C1 2 25 1 1 21 50
Sample 1BZ Sample 12 hours 18 C2 2 25 1 1 21 50
Sample 4AZ No Template 12 hours 19 C3 2 25 1 1 21 50
Sample 4BZ No Template 12 hours 19 C4 2 25 1 1 21 50
Sample 1AZ Sample 3 hours-Epicentre 20 C5 2 25 1 1 21 50
Sample 1BZ Sample 3 hours-Epicentre 20 C6 2 25 1 1 21 50
Sample 4AZ No Template 3 hours-Epicentre 21 C7 2 25 1 1 21 50
Sample 4BZ No Template 3 hours-Epicentre 21 C8 2 25 1 1 21 50
NTC None   22 F1 0 25 1 1 23 50

Results[edit]

CT Table[edit]

Sample No Condition CT PreRCA Sample Fold Diff Over PreRCA Conc. Difference
1AZ 12 hours 11.52 1A 7.3 153.3
4BZ 12 hours 11.52 4B 6.3 78.2
1BZ 12 hours 11.93 1B 5.9 57.7
4AZ 12 hours 12.07 4A 5.2 36.5
1BY 6 hours 12.38 1B 5.4 42.2
1AY 6 hours 13.26 1A 5.5 45.9
3BY 6 hours 14.35 3B 3.5 11.3
3AY 6 hours 14.59 3A 3.2 9.1
2BX 3 hours 15.16 2B 2.3 4.8
1BQ 3 hours-Epicentre 15.17 1B 2.6 6.1
2AX 3 hours 15.3 2A 2.1 4.3
1AQ 3 hours-Epicentre 15.31 1A 3.5 11.1
1AX 3 hours 15.42 1A 3.4 10.3
1BX 3 hours 15.46 1B 2.3 5.0
4BQ 3 hours-Epicentre 16.37 4B 1.4 2.7
4AQ 3 hours-Epicentre 17.07 4A 0.2 1.1
4A PreRCA 17.26 4A 0.0 1.0
2A PreRCA 17.39 2A 0.0 1.0
2B PreRCA 17.43 2B 0.0 1.0
3A PreRCA 17.77 3A 0.0 1.0
1B PreRCA 17.78 1B 0.0 1.0
4B PreRCA 17.81 4B 0.0 1.0
3B PreRCA 17.85 3B 0.0 1.0
1A PreRCA 18.78 1A 0.0 1.0