Daniel:Notebook/ComboLock/2016-9-30: Difference between revisions

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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
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| width="72" | Amount


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Revision as of 23:30, 3 October 2016

AmpR Test

Back to Calendar

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 Amount
    0 1 100000000000 1 pmol
    1 10 10000000000 100 fmol
    2 100 1.00E+09 10 fmol
    3 1000 1.00E+08 1 fmol
    4 10000 1.00E+07 100 amol
    5 100000 1.00E+06 10 amol

    Buffers

    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

    Protocol

    1. Latch and Padlock Hybridization
      1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
      2. Apply magnet for 30 sec and remove supernatant
      3. Add 2 uL oligo per sample to bead solution; incubate at RT for 5 min
      4. Apply magnet for 30 sec and remove supernatant
      5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
      6. Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube
      7. Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
      8. Chill on ice for 3 minutes
      9. Add sample to prepared strep beads; vortex to suspend
      10. Incubate for 30 min at 37C with intermittent mixing
      11. Apply magnet and remove supernatant
      12. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
      13. Repeat wash step above
      14. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
      15. Resuspend with 20 uL 1x Amp Ligase buffer
    2. Circularization
      1. Prepare 30 uL KLN mix
      2. Add 2 uL KLN mix to 20 uL of reaction
      3. Incubate at 55C overnight; continued tomorrow