Daniel:Notebook/ComboLock/2017-3-13: Difference between revisions
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[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
==Protocol== | ==Protocol-No Ligase== | ||
'''Sample Matrix''' | '''Sample Matrix''' | ||
Line 14: | Line 14: | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Sample 5 (AB) | | height="15" valign="bottom" | Sample 5 (AB) | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | Normal | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Sample 6 (AB) | | height="15" valign="bottom" | Sample 6 (AB) | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | No Ligase | ||
|} | |} | ||
Line 103: | Line 103: | ||
<li>Incubate at RT for 15 min</li> | <li>Incubate at RT for 15 min</li> | ||
<li>Heat kill enzyme by incubating at 65C for 10 minutes</li> | <li>Heat kill enzyme by incubating at 65C for 10 minutes</li> | ||
<li>Wash twice with 100 uL wash buffer</li> | |||
</ol> | </ol> | ||
****** | |||
<li>Elution</li> | |||
<ol type="A"> | |||
<li>Add 50 uL wash buffer to each sample</li> | |||
<li>Cook at 95C for 15 minutes</li></ol> | |||
****** | ****** | ||
<li>qPCR</li> | <li>qPCR</li> | ||
Line 109: | Line 115: | ||
<li>Make master mixes according to following recipes</li> | <li>Make master mixes according to following recipes</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#66CCFF;font-size:12pt;font-weight:bold" align="center" | |||
| width="220" height="30" | Reagent | |||
| width="85" | Single Rxn | |||
| width="85" | MMC1 (6.2X) | |||
| width="85" | MMC2 (6.2X) | |||
| width="85" | MMP (6.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Primer Pair | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | p24/p4RC | |||
| align="center" valign="bottom" | p6/p12RC | |||
| align="center" valign="bottom" | p24/p12RC | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | uL Fwd Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | uL Rev Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | uL nfH2O | |||
| align="center" align="center" valign="bottom" | 21 | |||
| align="center" align="center" valign="bottom" | 130.2 | |||
| align="center" align="center" valign="bottom" | 130.2 | |||
| align="center" align="center" valign="bottom" | 130.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | uL Kapa SYBR Fast | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 155 | |||
| align="center" align="center" valign="bottom" | 155 | |||
| align="center" align="center" valign="bottom" | 155 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 48 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 297.6 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 297.6 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 297.6 | |||
|} | |||
<li>Add 48 uL master mix to each well</li> | <li>Add 48 uL master mix to each well</li> | ||
<li>Add 2 uL sample according to plate layout</li> | <li>Add 2 uL sample according to plate layout</li> | ||
[[Image:PlateLayout- | [[Image:PlateLayout-03132017-BoltTest-NoLigase.png|600px]] | ||
*'''Note''': The parentheses in the descriptions indicate which sample was used | *'''Note''': The parentheses in the descriptions indicate which sample was used | ||
Line 142: | Line 199: | ||
===Results=== | ===Results=== | ||
<gallery perrow= | <gallery perrow=3 heights=300px widths=300px> | ||
File:|qPCR curves | File:20170313-qPCR-BoltTest-NoLigase.png|qPCR curves | ||
File:| | File:2017-03-13-BoltTest-NoLigase.png|Gel image-product targets | ||
File:| | File:2017-03-14-BoltTest-NoLigase-Purified.png|Gel image-product targets-Qiaquick purified | ||
</gallery> | </gallery> | ||
===Results-CANTC and Efficiency=== | |||
Take this with the [[Daniel:Notebook/ComboLock/2017-3-10|Friday results]] | |||
[[Image:20170313-BoltTest-NoLigase-SampleValues.png|600px]] | |||
[[Image:20170313-BoltTest-NoLigase-Efficiencies.png|600px]] | |||
The above graph (and Friday's, which is just this graph without the Sample-TR2 and No Ligase samples) had potentially wrong calculations for efficiency, since they only calculated the amount in the qPCR reaction (2 uL), which is a small fraction of the amount eluted (50 uL). | |||
[[Image:20170313-BoltTest-NoLigase-Efficiencies-Corrected.png|600px]] | |||
[[Category:ComboLock]] [[Category:20170308]] | [[Category:ComboLock]] [[Category:20170308]] |
Latest revision as of 20:25, 14 March 2017
Bolt Test (Started Wednesday March 8)[edit]
Protocol-No Ligase[edit]
Sample Matrix
Sample | Condition |
Sample 5 (AB) | Normal |
Sample 6 (AB) | No Ligase |
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- C Probe Hybridization
- Combine following into a 0.2 mL tube
- 4 uL each PCCA-03 and PCCB-03 (10 uM each)
- 12 uL wash buffer
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Repeat wash step above
Protocol-Part 2-LB hybridization & ligation[edit]
- Bolt and Latch Hybridization/Ligation
- Set up the following master mix
- Add 19 uL probe mix to each sample
- Incubate at 55C for 10 minutes
- Incubate at 25C for 10 minutes
- Lower the temperature to 20C; wait 30 seconds then take off incubator
- Add 1 uL T4 Ligase to each reaction
- Incubate at RT for 15 min
- Heat kill enzyme by incubating at 65C for 10 minutes
- Wash twice with 100 uL wash buffer
- Elution
- Add 50 uL wash buffer to each sample
- Cook at 95C for 15 minutes
- qPCR
- Make master mixes according to following recipes
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-03132017-BoltTest-NoLigase.png
- Note: The parentheses in the descriptions indicate which sample was used
- Note:The colors are according to master mix, targeting either C1 (blue), C2 (red), or product (green)
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 360 uL TBE, 90 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (4.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 16.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 4.2 |
Lock Oligo | 10 uM | 10 pmol | 1 | 4.2 |
nfH2O | NA | NA | 13 | 54.6 |
Total | 19 | 79.8 |
Reagent | Single Rxn | MMC1 (6.2X) | MMC2 (6.2X) | MMP (6.2X) |
Primer Pair | NA | p24/p4RC | p6/p12RC | p24/p12RC |
uL Fwd Primer (10 uM) | 1 | 6.2 | 6.2 | 6.2 |
uL Rev Primer (10 uM) | 1 | 6.2 | 6.2 | 6.2 |
uL nfH2O | 21 | 130.2 | 130.2 | 130.2 |
uL Kapa SYBR Fast | 25 | 155 | 155 | 155 |
Total | 48 | 297.6 | 297.6 | 297.6 |
Results[edit]
- 20170313-qPCR-BoltTest-NoLigase.png
qPCR curves
- 2017-03-13-BoltTest-NoLigase.png
Gel image-product targets
- 2017-03-14-BoltTest-NoLigase-Purified.png
Gel image-product targets-Qiaquick purified
Results-CANTC and Efficiency[edit]
Take this with the Friday results
File:20170313-BoltTest-NoLigase-SampleValues.png
File:20170313-BoltTest-NoLigase-Efficiencies.png
The above graph (and Friday's, which is just this graph without the Sample-TR2 and No Ligase samples) had potentially wrong calculations for efficiency, since they only calculated the amount in the qPCR reaction (2 uL), which is a small fraction of the amount eluted (50 uL).