Daniel:Notebook/ComboLock/2016-8-16: Difference between revisions
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<li>Phosphorylation</li> | <li>Phosphorylation</li> | ||
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</li> | <li>Incubate at 37C for 30 min</li> | ||
</ol> | |||
<li>Ligation</li> | |||
<ol type="A"> | |||
<li>Set up 2 reactions with the following reagents</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | |||
| width="162" height="45" | Reagent | |||
| width="71" | Stock Conc | |||
| width="79" | Final Conc./Amount | |||
| width="65" | uL added | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | AmpLigase Reaction Buffer | |||
| align="center" | 10X | |||
| align="center" | 1X | |||
| align="center" align="center" | 4 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Latch Oligo | |||
| align="center" | 100 uM | |||
| align="center" | 500 umol | |||
| align="center" align="center" | 5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 5' Amplicon Oligo | |||
| align="center" | 100 uM | |||
| align="center" | 500 umol | |||
| align="center" align="center" | 5 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase | |||
| align="center" | 5 U/uL | |||
| align="center" | 5 U | |||
| align="center" align="center" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phosphate Reaction | |||
| align="center" | NA | |||
| align="center" | NA | |||
| align="center" align="center" | 10 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" | NA | |||
| align="center" | NA | |||
| align="center" align="center" | 25 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
|style="font-weight:bold" align="center" align="center" | 50 | |||
|} | |||
<li>Add amplicons according to following table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="45" | Sample | |||
| width="65" | PCAmp1 | |||
| width="65" | PCAmp3 | |||
| width="65" | LatchX2 | |||
| width="65" | LatchX3 | |||
|- style="font-size:12pt" | |||
| height="15" align="center" | AmpliconX1 | |||
| align="center" | X | |||
| align="center" valign="bottom" | | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| height="15" | AmpliconX3 | |||
| align="center" | | |||
| X | |||
| align="center" | | |||
| X | |||
|} | |||
<li> | |||
</ol> |
Revision as of 17:25, 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
- Add amplicons according to following table
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 |