Daniel:Notebook/ComboLock/2016-9-29: Difference between revisions

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>Djacobse
>Djacobse
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File:2016-09-29-qPCR-Primer12RC.png|Gel Image
File:2016-09-29-qPCR-Primer12RC.png|Gel Image
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===Discussion===
As I expected there is no extra product (or very little) being formed.


[[Category:ComboLock]] [[Category:20160928]]
[[Category:ComboLock]] [[Category:20160928]]

Revision as of 20:19, 30 September 2016

Positive Control Amplicon Production (PCAmp4; Started yesterday)

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Protocol

  1. Size Selection
    1. Centrifuge at 12000 rpm for 1.5 minutes
    2. Extract the supernatant and place into NanoSep column
    3. Centrifuge at 12000 rpm for 1.5 minutes
  2. Ethanol Precipitation
    1. Move sample to a fresh 2 mL tube
    2. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue
    3. Store at for 6 hours
    4. Pellet by centrifuging for 25 minutes at 12000rpm at 4C
    5. Remove supernatant and add 750 uL chilled 70% EtOH
    6. Centrifuge for 12 minutes at 12000rpm at 4C
    7. Remove supernatant and spin in vacuum centrifuge for 10 minutes
    8. Add 50 uL nfH2O and resuspend every tube in series;
    9. Measure final concentration in nanodrop

Results

Sample ng/uL ssDNA
C4-C2 Amplicon (Final) 78.3

AmpF Test

Since I'm testing whether the AmpF primer (which has LinkerR that matches to primer2 and therefore has a potential off-target binding) creates the wrong product or not. To test this I'll be using the PCR primer Primer12-RC, which should be a forward primer for amplifying off the circular target. In conjuction with AmpR6.3, this should produce a 157bp product. Let's see what happens.

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 primer12RC 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1A 1.00E+09 A1 37 2 25 0.5 5 17.5 50
      Sample 1B 1.00E+09 A2 37 2 25 0.5 5 17.5 50
      Sample 1C 1.00E+09 A3 37 2 25 0.5 5 17.5 50
      NTC 0 A4 37 0 25 0.5 5 19.5 50


    3. Make the following master mix
      1. 71.75 uL nfH2O
      2. 2.05 uL Primer12RC
      3. 20.5 uL AmpR6.3-34
      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

    As I expected there is no extra product (or very little) being formed.