Daniel:Notebook/ComboLock/2016-9-30

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Revision as of 22:01, 30 September 2016 by >Djacobse (→‎Discussion)
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AmpR Test

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Yesterday I tested the AmpR primer (the names are actually confusing, the name is the primer I'm NOT using. So yesterday I amplified off the circularized product using AmpR (which binds to the LinkerL region of the circularized product) and primer12RC, which binds to the amplified product. Now I'm going to test the amplification of using the AmpF primer. If AmpF is the culprit (which is what I expect) then I should see off-target binding. If the off-target products require both AmpF and AmpR, I shouldn't see off-target product.

Protocol

  1. qPCR
    1. Follow reaction table below to set up 4 reactions
    2. Sample Molecule Count Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF6.4Sol 100 uM primer2 H2O Total Volume (uL)
      Sample 0A 1.00E+10 A1 NA 2 25 0.5 0.5 22 50
      Sample 0B 1.00E+10 A2 NA 2 25 0.5 0.5 22 50
      Sample 0C 1.00E+10 A3 NA 2 25 0.5 0.5 22 50
      NTC 0 A4 NA 0 25 0.5 0.5 24 50
    3. Make the following master mix
      1. 90.2 uL nfH2O
      2. 2.05 uL Primer2
      3. 2.05 uL AmpF6.4Sol
      4. 102.5 uL SYBR Fast
    4. Aliquot 48 uL master mix into each well
    5. Add 2 uL sample to each well
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x26
      7. 72C 2 min
      8. 16C hold

    Results

    Discussion

    Well, this pretty much confirms it. AmpF is the problem and is producing off-target products. Note that the expected product is the 168 bp band, but a primer dimer caused by the off-target binding would produce a product of approximately 75bp. We see a clear band at 75 bp, meaning that the AmpF-primer2 binding is likely too strong. Hopefully with changing primer2 to primer24 (see PCAmp4 production) for further details.

    Positive Control Amplicon 4 Test

    PCAmp4 uses the primer24 adapter instead of primer2 and should not have the same problems in amplification that the primer2-based system has. See Wednesday for the production of PCAmp4 (C4-C2).

    Dilutions

    Tube No Dilution Molecules Available
    0 1 100000000000
    1 10 10000000000
    2 100 1.E+09
    3 1000 1.E+08
    4 10000 1.E+07
    5 100000 1.E+06

    KLN Mix

    Reagent Stock uL added
    Amp Ligase Buffer 10X 3
    dNTPs 100 uM 3
    Hemo Klentaq NA 6
    Amp Ligase 5 U/uL 3
    nfH2O NA 15