Daniel:Notebook/ComboLock/2016-8-23: Difference between revisions
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=Positive Control Amplicon Test 2 (Started [[Daniel:Notebook/ComboLock/2016-8- | =Positive Control Amplicon Test 2 (Started [[Daniel:Notebook/ComboLock/2016-8-22|yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
Line 189: | Line 189: | ||
<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px> | ||
File:|qPCR Curve | File:20160823-qPCRCurve-X2.png|X2 qPCR Curve | ||
File:|Gel Image | File:2016-08-23-PosConX2.png|X2 Gel Image | ||
File:20160823-qPCRCurve-X3.png|X3 qPCR Curve | |||
File:2016-08-23-PosConX3.png|X3 Gel Image | |||
</gallery> | </gallery> | ||
[[Category:ComboLock]] [[Category: | =Positive Control Amplicon Production= | ||
==Protocol== | |||
<ol> | |||
<li>Phosphorylation</li> | |||
<ol type="A"> | |||
<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> | |||
</ol> | |||
<li>Ligation</li> | |||
<ol type="A"> | |||
<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> | |||
{| 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>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 55C for 2 hours</li> | |||
<li>Heat to 95 C to denature dsDNA | |||
<li>Purify with ssDNA column</li> | |||
</ol> | |||
<li>ssDNA Column</li> | |||
<ol type="A"> | |||
<li>Add 100 uL '''Binding Buffer''' to the sample; mix well</li> | |||
<li>Transfer to '''IIC Column''' and centrifuge at 14000 rpm for 1 minute; '''SAVE THE FLOW THROUGH'''</li> | |||
<li>Add 150 uL 100% EtOH to flow through; mix well</li> | |||
<li>Transfer to '''IC Column''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> | |||
<li>Add 400 uL '''Prep Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> | |||
<li>Add 700 uL '''Wash Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> | |||
<li>Add 400 uL '''Wash Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> | |||
<li>Centrifuge empty column at 14000 rpm for 2 minutes</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> | |||
</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" | 287.6 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | C3-C2 Amplicon | |||
| align="center" align="center" valign="bottom" | 331.6 | |||
|} | |||
==Protocol (Part 2)== | |||
<ol start="4"> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 32 uL TBE and 8 uL 6x Gel Loading dye</li> | |||
<li>Aliquot 10 uL for each sample to parafilm</li> | |||
<li>Add 2 uL of diluted sample/ladder to the drops</li> | |||
<li>Mix with the loading pipette and add 10 uL to each lane</li> | |||
<li>Run for 25 min at 250V</li> | |||
<li>Stain with 3 uL SYBR Gold for 3 min</li> | |||
<li>Rinse and image (see gallery below)</li> | |||
</ol></ol> | |||
[[Image:2016-08-23-PosConAmplicon.png|500px]] | |||
<ol start="5"> | |||
<li>Size Select</li> | |||
<ol type="A"> | |||
<li>For sample lanes, mix 16 uL sample, 14 uL 6X loading dye and 50 uL TBE (80 uL total)</li> | |||
<li>For ladder lanes, mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)</li> | |||
<li>Add 20 uL to each lane (2 gels total, 1 per sample)</li> | |||
<li>Run gel for 25 minutes at 250V</li> | |||
<li>While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube</li> | |||
<li>Stain gel for 3 minutes with 3 uL SYBR gold</li> | |||
<li>Image in gel doc</li> | |||
<li>Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)</li> | |||
<li>Image in gel doc post-extraction</li> | |||
<li>Centrifuge tubes at 12000rpm for 1.5 minutes</li> | |||
<li>Discard 0.5 mL tube and resuspend in 500 uL TE buffer</li> | |||
<li>Incubate at 37C for 1 hour with vigorous shaker</li> | |||
<li>Centrifuge at 12000 rpm for 1.5 minutes</li> | |||
<li>Extract the supernatant and place into NanoSep column</li> | |||
<li>Centrifuge at 12000 rpm for 1.5 minutes</li> | |||
</ol> | |||
<li>Ethanol Precipitation</li> | |||
<ol type="A"> | |||
<li>Move sample to a fresh 2 mL tube</li> | |||
<li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue</li> | |||
<li>Store at -80C overnight, continued [[Daniel:Notebook/ComboLock/2016-8-24|tomorrow]]</li> | |||
</ol></ol> | |||
[[Category:ComboLock]] [[Category:20160822]] [[Category:20160823]] |
Latest revision as of 22:42, 26 August 2016
Positive Control Amplicon Test 2 (Started yesterday)[edit]
Protocol[edit]
- 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
- 20160823-qPCRCurve-X2.png
X2 qPCR Curve
- 2016-08-23-PosConX2.png
X2 Gel Image
- 20160823-qPCRCurve-X3.png
X3 qPCR Curve
- 2016-08-23-PosConX3.png
X3 Gel Image
- 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
- TBE Gel
- Mix 32 uL TBE and 8 uL 6x Gel Loading dye
- Aliquot 10 uL for each sample to parafilm
- Add 2 uL of diluted 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)
- Size Select
- For sample lanes, mix 16 uL sample, 14 uL 6X loading dye and 50 uL TBE (80 uL total)
- For ladder lanes, mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)
- Add 20 uL to each lane (2 gels total, 1 per sample)
- Run gel for 25 minutes at 250V
- While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube
- Stain gel for 3 minutes with 3 uL SYBR gold
- Image in gel doc
- Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)
- Image in gel doc post-extraction
- Centrifuge tubes at 12000rpm for 1.5 minutes
- Discard 0.5 mL tube and resuspend in 500 uL TE buffer
- Incubate at 37C for 1 hour with vigorous shaker
- Centrifuge at 12000 rpm for 1.5 minutes
- Extract the supernatant and place into NanoSep column
- Centrifuge at 12000 rpm for 1.5 minutes
- Ethanol Precipitation
- Move sample to a fresh 2 mL tube
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue
- Store at -80C overnight, continued tomorrow
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[edit]
Positive Control Amplicon Production[edit]
Protocol[edit]
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[edit]
Sample | ng/uL ssDNA |
C1-C2 Amplicon | 287.6 |
C3-C2 Amplicon | 331.6 |
Protocol (Part 2)[edit]
File:2016-08-23-PosConAmplicon.png