Daniel:Notebook/ComboLock/2016-8-16

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Positive Control Amplicon Production[edit]

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Theory[edit]

The C1 and C3 amplicons

C1 Amplicon (PCAmp1): 5-ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG-3

C2 Amplicon (PCAmp2): 5-GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/-3

C3 Amplicon (PCAmp3): 5-ACGGCGGACCTCGCACGGCTGCCAACCCGTGGACGGTCGCGTTCGATGCGTC-3

Reaction Trimers:

Note that the | symbol denotes the break between the two amplicons and the space on the latches is just for visualization

LatchX2-Rev                              3-GGCACCTGCCAGCGCAAGTGATTTAC CGGGCATAGCCGCTACGCACTAGCCC-5     
PCAmp1|PCAmp2  5-ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG|GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/
LatchX3-Rev                              3-GGCACCTGCCAGCGCAAGCTACGCAG CGGGCATAGCCGCTACGCACTAGCCC-5
PCAmp3|PCAmp2  5-ACGGCGGACCTCGCACGGCTGCCAACCCGTGGACGGTCGCGTTCGATGCGTC|GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/

Protocol[edit]

To complete the ligation reaction it is necessary that the oligo on the 3' end of the amplicon have a phosphate group on its 5' end. This requires a T4 Polynucleotide Kinase reaction (or ordering an oligo with a 5' phosphate). After the addition of the 5' phosphate I will incubate the oligos together in equal molar ratios to and add ligase to perform the ligation reaction.

  1. Prep
    1. Resuspend each amplicon to 100 uM according to table
    2. Sequence uL nfH2O
      PCAmp1 650
      PCAmp2 753
      PCAmp3 699
      LatchX2 631
      LatchX3 737
  2. Phosphorylation
    1. In a 0.2 mL tube, add ingredients according to table
    2. Reagent Stock Conc Final Conc./Amount uL added
      AmpLigase Reaction Buffer 10X 1X 2
      ATP 10 mM 1 mM 2
      PCAmp2 100 uM 1 nmol total 10
      T4 DNA Kinase 10 U/uL 10 U 1
      nfH2O NA NA 5
      Total     20
    3. Incubate at 37C for 30 min
  3. Ligation
    1. Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)
    2. Reagent Stock Conc Final Conc./Amount uL added
      AmpLigase Reaction Buffer 10X 1X 4
      Latch Oligo 100 uM 500 umol 5
      5' Amplicon Oligo 100 uM 500 umol 5
      Amp Ligase 5 U/uL 5 U 1
      Phosphate Reaction NA NA 10
      nfH2O NA NA 25
      Total     50
    3. Add amplicons according to following table
    4. Sample PCAmp1 PCAmp3 LatchX2 LatchX3
      AmpliconX1 X   X  
      AmpliconX3   X   X
    5. Heat reaction to 95C for 5 min
    6. Lower temp to 55C
    7. Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix
    8. Incubate at 55C for 2 hours
    9. Heat to 95 C to denature dsDNA
    10. Purify with ssDNA column
  4. ssDNA Column
    1. Add 100 uL Binding Buffer to the sample; mix well
    2. Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
    3. Add 150 uL 100% EtOH to flow through; mix well
    4. Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
    5. Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    6. Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    7. Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    8. Centrifuge empty column at 14000 rpm for 2 minutes
    9. Transfer to empty 1.5mL centrifuge tube (low bind)
    10. Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute

    Nanodrop Results[edit]

    Sample ng/uL ssDNA
    C1-C2 Amplicon 293.7
    C3-C2 Amplicon 326.4

    Protocol (Part 2)[edit]

    1. TBU gel
      1. Prerun gel for 15 minutes at 250V
      2. Mix 9 uL TBE, 10 uL 2X Buffer, 1 uL sample for 10 bp ladder, C1-C2 amplicon, and C3-C2 amplicon
      3. Remove urea from wells by pipetting up and down with a 200 uL pipette
      4. Add 20 uL to each lane
      5. Run gel for 25 min at 250V
      6. Stain with 3 uL SYBR Gold for 3 min
      7. Rinse and image (see gallery below)

    Results are inconclusive, I'm going to try a TBE gel with the 25bp ladder, which is generally easier to read than the 10bp ladder

    1. TBE Gel
      1. Mix 36 uL TBE and 8 uL 6x Gel Loading dye
      2. Aliquot 10 uL for each sample to parafilm
      3. Add 1 uL of sample/ladder to the drops
      4. Mix with the loading pipette and add 10 uL to each lane
      5. Run for 25 min at 250V
      6. Stain with 3 uL SYBR Gold for 3 min
      7. Rinse and image (see gallery below)

    Gel Imaging Results[edit]

    Protocol (Part 3)[edit]

    Since the TBE gel showed a smear at the correct range (104 bp, but with a lot of DNA and biotin attached it runs funny), I'll be moving on to a size select and purification.

    1. Size select gel
      1. Mix 40 uL TBE, 10 uL loading dye, and 10 uL sample for C1-C2 and C3-C2
      2. Mix 23 uL TBE, 5 uL loading dye, and 2 uL ladder
      3. Add 30 uL to each lane (2 lanes for C1-C2 and C3-C2)
      4. Run the gel at 250V for 25 min
      5. Stain with 3 uL SYBR gold for 3 min
      6. Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube
      7. Shred the gel by centrifuging at 14000rpm for 1 min 30 sec

      As you can see from the gel images, there was too much DNA on the gel and it ran funny. Besides, the caps fell off in the centrifuge.