Daniel:Notebook/ComboLock/2016-8-16
Positive Control Amplicon Production
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.
- Prep
- Resuspend each amplicon to 100 uM according to table
- Phosphorylation
- In a 0.2 mL tube, add ingredients according to table
- Incubate at 37C for 30 min
- Ligation
- Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)
- Add amplicons according to following table
- Heat reaction to 95C for 5 min
- Lower temp to 55C
- Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix
- Incubate at 55C for 2 hours
- Heat to 95 C to denature dsDNA
- Purify with ssDNA column
- ssDNA Column
- Add 100 uL Binding Buffer to the sample; mix well
- Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
- Add 150 uL 100% EtOH to flow through; mix well
- Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Centrifuge empty column at 14000 rpm for 2 minutes
- Transfer to empty 1.5mL centrifuge tube (low bind)
- Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute
- TBU gel
- Prerun gel for 15 minutes at 250V
- Mix 9 uL TBE, 10 uL 2X Buffer, 1 uL sample for 10 bp ladder, C1-C2 amplicon, and C3-C2 amplicon
- Remove urea from wells by pipetting up and down with a 200 uL pipette
- Add 20 uL to each lane
- Run gel for 25 min at 250V
- Stain with 3 uL SYBR Gold for 3 min
- Rinse and image (see gallery below)
- TBE Gel
- Mix 36 uL TBE and 8 uL 6x Gel Loading dye
- Aliquot 10 uL for each sample to parafilm
- Add 1 uL of sample/ladder to the drops
- Mix with the loading pipette and add 10 uL to each lane
- Run for 25 min at 250V
- Stain with 3 uL SYBR Gold for 3 min
- Rinse and image (see gallery below)
- 2016-08-16-PosControlAmplicon-TBU.png
TBU Gel Image
- 2016-08-16-PosControlAmplicon-TBE.png
TBE Gel Image
Sequence | uL nfH2O |
PCAmp1 | 650 |
PCAmp2 | 753 |
PCAmp3 | 699 |
LatchX2 | 631 |
LatchX3 | 737 |
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 |
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 |
Sample | PCAmp1 | PCAmp3 | LatchX2 | LatchX3 |
AmpliconX1 | X | X | ||
AmpliconX3 | X | X |
Nanodrop Results
Sample | ng/uL ssDNA |
C1-C2 Amplicon | 293.7 |
C3-C2 Amplicon | 326.4 |
Protocol (Part 2)
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