Daniel:Notebook/ComboLock/2016-5-25

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Revision as of 23:03, 25 May 2016 by >Djacobse (→‎Prep for VIM Antibody Test)
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C Probe Hybridization test (VIM, Started 5/23/2016)

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From yesterday's results, I'm going to try the qPCR again. I saved the samples in the 4C, so I just went straight to the qPCR. Hopefully this time I will get more conclusive evidence one way or the other.

Protocol

  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


Results

Row Sample Cq
A01 Sample 1 (C1) 15.16
B01 Sample 2 (C2) 13.55
C01 Sample 3 (C1+C2) 13.45
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 Results2 20160525 VIM.png

Discussion

Once again all the negative controls failed and so did the positive control. However, this time all 3 samples amplified correctly. I'm inclined therefore to call this a successful run, which means I should try the other samples.

Prep for VIM Antibody Test

Tomorrow I plan on beginning the anitbody prep for testing VIM. Today there is some prep work to be done.

  1. Prep 100 mL of sodium phosphate buffer
    1. Make 100 mL of NaH2PO4 (monobasic) by adding 13.8g to nfH2O
    2. Make 100 mL of Na2H2PO4 (dibasic) by adding 14.2g to nfH2O
    3. Mix 28 mL of monobasic and 72 mL of dibasic to make 1 M (buffer pH 7.2)
    4. Directions from Cold Spring Harbor
  2. Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer