Daniel:Notebook/ComboLock/2016-5-24

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C Probe Hybridization Test (VIM, Prep yesterday)[edit]

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Protocol[edit]

Sample Matrix[edit]

Sample VIM-C1 (1 uL) VIM-C2 (1 uL)
Sample 1 X  
Sample 2   X
Sample 3 X X
Sample 4    
  1. Hybridization (Lab)
    1. Split cells into 4 samples (1.25 mL methanol each)
    2. Pellet cells via centrifugation at 600g for 3 min
    3. Put 1 uL C1 Probe and 1 uL C2 probe into separate 0.2 mL tubes
    4. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    5. Add probes to hybridization buffer for final concentration of 100 nM (0.5 uL/probe/sample using 0.5mL hybridization buffer)
    6. Incubate probes at 40C for 1 hour with vigorous agitation
    7. Wash three times with 0.5 mL wash buffer and pelleting at 600xg for 3 min
  2. qPCR
    1. Set up reaction according to table
    2. Sample Lane Cell Sample Cells 2X Kapa SYBR qPCR MM 100 uM Primer2 100 uM Primer4-RC 100 uM Primer6 100 uM Primer12-RC H2O Total Volume (uL)
      Sample 1 (C1) A1 S1 1 25 0.5 0.5 0 0 23 50
      Sample 2 (C2) A2 S2 1 25 0 0 0.5 0.5 23 50
      Sample 3 (C1+C2) A3 S3 1 25 0.5 0.5 0.5 0.5 22 50
      Sample 4 (No Probe) A4 S4 1 25 0.5 0.5 0.5 0.5 22 50
      No Template A5 NA 0 25 0.5 0.5 0.5 0.5 23 50
      Mismatch Primers A6 S3 1 25 0.5 0 0.5 0 23 50
      No Primers A7 S3 1 25 0 0 0 0 24 50
      Primers + C Probes A8 NA 1 25 0.5 0.5 0.5 0.5 22 50
    3. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x20
      7. 72C 2 min
      8. 16C hold

Buffers[edit]

  1. Wash Buffer: 1X PBS, 0.1% Tween-20, 4 U/mL RNasin
  2. Hybridization Buffer: 1X SSC, 20mM RVC, 40 U/mL RNasin, 0.1% Tween 20, 100 ug/mL salmon sperm DNA

qPCR Results[edit]

Row Sample Cq
A01 Sample 1 (C1) N/A
B01 Sample 2 (C2) 13.1
C01 Sample 3 (C1+C2) 12.99
D01 Sample 4 (No Probe) N/A
E01 No Template N/A
F01 Mismatch Primers N/A
G01 No Primers N/A
H01 Primers + C Probes N/A

File:CProbeTest Results.png

Discussion[edit]

The negative controls were all negative, which is good. However, the positive control showed no results and only 2 of 3 samples amplified. I think it would be best to repeat the experiment tomorrow.