Daniel:Notebook/ComboLock/2016-8-16: Difference between revisions

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<li>Add amplicons according to following table</li>
<li>Add amplicons according to following table (DO NOT ADD LIGASE YET)</li>
{| 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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<li>
<li>Heat reaction to 95C for 5 min</li>
<li>Lower temp to 55C</li>
<li>Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix</li>
<li>Incubate at 35C for 2 hours</li>
<li>Purify with DNA column; elute with 30 uL EB</li>
</ol>
</ol>

Revision as of 17:36, 16 August 2016

Positive Control Amplicon Production

Back to Calendar

Theory

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

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
    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 (DO NOT ADD LIGASE YET)
    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 35C for 2 hours
    9. Purify with DNA column; elute with 30 uL EB