Daniel:Notebook/ComboLock/2016-8-23: Difference between revisions
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>Djacobse |
>Djacobse |
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<ol type="A"> | <ol type="A"> | ||
<li>In a 0.2 mL tube, add ingredients according to table</li> | <li>In a 0.2 mL tube, add ingredients according to table</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="153" height="36" | Reagent | |||
| width="73" | Stock Conc | |||
| width="90" | 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" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | ATP | |||
| align="center" | 10 mM | |||
| align="center" | 1 mM | |||
| align="center" align="center" | 2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | PCAmp2 | |||
| align="center" | 100 uM | |||
| align="center" | 1 nmol total | |||
| align="center" align="center" | 10 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | T4 DNA Kinase | |||
| align="center" | 10 U/uL | |||
| align="center" | 10 U | |||
| align="center" align="center" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" | NA | |||
| align="center" | NA | |||
| align="center" align="center" | 5 | |||
|- 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" | 20 | |||
|} | |||
<li>Incubate at 37C for 30 min</li> | <li>Incubate at 37C for 30 min</li> | ||
Line 205: | Line 250: | ||
<ol type="A"> | <ol type="A"> | ||
<li>Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)</li> | <li>Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)</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> | <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>Heat reaction to 95C for 5 min</li> | <li>Heat reaction to 95C for 5 min</li> | ||
<li>Lower temp to 55C</li> | <li>Lower temp to 55C</li> |
Revision as of 16:53, 23 August 2016
Positive Control Amplicon Test 2 (Started yesterday)
Protocol
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table
- Master Mix
- 277.5 uL KAPA SyberFast MM
- 194.25 uL nfH2O
- 5.55 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- 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
Sample | Name | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | X2-1X | A1 | 33 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | X2-5X | B1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | X2-10X | C1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | X2-50X | D1 | 36 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | X3-1X | E1 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | X3-5X | F1 | 38 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | X3-10X | G1 | 39 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8 | X3-50X | H1 | 40 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | X2Flip | A12 | 41 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10 | X3Flip | B12 | 42 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | PCR NTC | C12 | 43 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |
Results
Positive Control Amplicon Production
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 |