Daniel:Notebook/ComboLock/2016-8-16: Difference between revisions
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<li>Transfer to empty 1.5mL centrifuge tube (low bind)</li> | <li>Transfer to empty 1.5mL centrifuge tube (low bind)</li> | ||
<li>Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute</li> | <li>Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute</li> | ||
</ol> | |||
===Nanodrop Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4BD97;font-size:12pt;font-weight:bold" align="center" | |||
| width="103" height="27" | Sample | |||
| width="77" | ng/uL ssDNA | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | C1-C2 Amplicon | |||
| align="center" align="center" valign="bottom" | 293.7 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | C3-C2 Amplicon | |||
| align="center" align="center" valign="bottom" | 326.4 | |||
|} | |||
==Protocol (Part 2)== | |||
<ol start="5"> | |||
<li>TBU gel</li> | |||
<ol type="A"> | |||
<li>Mix 9 uL TBE, 10 uL 2X Buffer, 1 uL sample for 10 bp ladder, C1-C2 amplicon, and C3-C2 amplicon</li> | |||
<li>Add 20 uL to each lane</li> | |||
<li>Run gel for 25 min at 250V</li> | |||
<li>Stain with 3 uL SYBR Gold for 3 min</li> | |||
<li>Rinse and image</li> | |||
[[Image:|500px]] | |||
</ol> | |||
<li>TBE Gel</li> | |||
</ol> | </ol> |
Revision as of 23:04, 16 August 2016
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
- Mix 9 uL TBE, 10 uL 2X Buffer, 1 uL sample for 10 bp ladder, C1-C2 amplicon, and C3-C2 amplicon
- 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 [[Image:|500px]]
- TBE Gel
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 |