Daniel:Notebook/ComboLock/2016-11-4: Difference between revisions
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>Djacobse |
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(14 intermediate revisions by the same user not shown) | |||
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<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Make master mix according to following recipe</li> | <li>Make master mix according to following recipe</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> uL nfH2O</li> | <li>233.1 uL nfH2O</li> | ||
<li> | <li>277.5 uL SYBR Fast</li> | ||
<li> | <li>11.1 uL 10 uM AmpF</li> | ||
</ol> | </ol> | ||
<li>Add 47 uL master mix to each well</li> | <li>Add 47 uL master mix to each well</li> | ||
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in following section</li> | |||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
<ol type="a"> | <ol type="a"> | ||
Line 29: | Line 29: | ||
<li>TBE Gel</li> | <li>TBE Gel</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Mix | <li>Mix 120 uL TBE, 30 uL 6x loading dye</li> | ||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | ||
<li>Add 2 uL of sample or ladder to correct drop</li> | <li>Add 2 uL of sample or ladder to correct drop</li> | ||
Line 186: | Line 186: | ||
|} | |} | ||
===Results=== | |||
<gallery perrow=3 heights=300px widths=300px> | |||
File:20161104-qPCR-AntibodyTest.png|qPCR curve | |||
File:2016-11-04-AntibodyTest-Gel1.png|Gel Image 1 | |||
File:2016-11-04-AntibodyTest-Gel2.png|Gel Image 2 | |||
</gallery> | |||
==Testing== | |||
Doing some QC control on the antibody experiment to figure out what went wrong. The first thing to test is the BSA Ab-oligo complexes. | |||
<ol> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<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:#FABF8F;font-size:12pt;font-weight:bold" align="center" | |||
| width="105" height="30" | Master Mix | |||
| width="65" | No. Samples | |||
| width="100" | Primers | |||
| width="120" | uL Reverse Primer (10 uM) | |||
| width="120" | uL Forward Primer (10 uM) | |||
| width="85" | uL Kapa SYBR Fast | |||
| width="85" | uL nfH2O | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | PA | |||
| align="center" align="center" valign="bottom" | 10 | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
| align="center" align="center" valign="bottom" | 10.1 | |||
| align="center" align="center" valign="bottom" | 10.1 | |||
| align="center" align="center" valign="bottom" | 252.5 | |||
| align="center" align="center" valign="bottom" | 212.1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | PB | |||
| align="center" align="center" valign="bottom" | 2 | |||
|style="font-weight:bold" align="center" | p6/p12RC | |||
| align="center" align="center" valign="bottom" | 2.1 | |||
| align="center" align="center" valign="bottom" | 2.1 | |||
| align="center" align="center" valign="bottom" | 52.5 | |||
| align="center" align="center" valign="bottom" | 44.1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Nonsense A | |||
| align="center" align="center" valign="bottom" | 2 | |||
|style="font-weight:bold" align="center" | p24/p12rc | |||
| align="center" align="center" valign="bottom" | 2.1 | |||
| align="center" align="center" valign="bottom" | 2.1 | |||
| align="center" align="center" valign="bottom" | 52.5 | |||
| align="center" align="center" valign="bottom" | 44.1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Nonsense B | |||
| align="center" align="center" valign="bottom" | 2 | |||
|style="font-weight:bold" align="center" valign="bottom" | p6/p4RC | |||
| align="center" align="center" valign="bottom" | 2.1 | |||
| align="center" align="center" valign="bottom" | 2.1 | |||
| align="center" align="center" valign="bottom" | 52.5 | |||
| align="center" align="center" valign="bottom" | 44.1 | |||
|} | |||
<li>Add 47 uL master mix to each well</li> | |||
<li>Add 2 uL sample according to table in following section</li> | |||
<li>qPCR Cycles</li> | |||
<ol type="a"> | |||
<li>95C 3 min</li> | |||
<li>95C 3 sec</li> | |||
<li>55C 30 sec</li> | |||
<li>72C 20 sec</li> | |||
<li>plate read</li> | |||
<li>goto b x24</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 160 uL TBE, 40 uL 6x loading dye</li> | |||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | |||
<li>Add 2 uL of sample or ladder to correct drop</li> | |||
<li>Load 10 uL in to well</li> | |||
<li>Run gel for 22 minutes at 250V</li> | |||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | |||
<li>Rinse gel and image in gel doc</li> | |||
</ol></ol> | |||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:20161104-qPCR-QC-AntibodyTest-PosNeg.png|qPCR curve comparing positive controls (BSA) vs. negative controls (NTC) | |||
File:20161104-qPCR-QC-AntibodyTest-TimeCourse.png|Time course comparison (negative controls included) | |||
File:2016-11-04-AntibodyTest-QC-Gel1.png|Gel Image 1 | |||
File:2016-11-04-AntibodyTest-QC-Gel2.png|Gel Image 2 | |||
</gallery> | |||
===qPCR Sample Table=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="105" height="45" | Sample | |||
| width="105" | Primers | |||
| width="85" | Primer Target | |||
| width="65" | Time Point | |||
| width="65" | Lane | |||
| width="65" | Sample Vol (uL) | |||
| width="105" | 2X Kapa SYBR qPCR MM | |||
| width="85" | 10 uM Forward Primer | |||
| width="85" | 10 uM Reverse Primer | |||
| width="65" | H2O | |||
| width="105" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | BSA-Ab | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" | PA | |||
|style="font-weight:bold" align="center" | NA | |||
| align="center" | A1 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | BSA-Ab | |||
|style="font-weight:bold" align="center" | p6/p12RC | |||
|style="font-weight:bold" align="center" | PB | |||
|style="font-weight:bold" align="center" | NA | |||
| align="center" | A2 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | BSA-Ab | |||
|style="font-weight:bold" align="center" | p24/p12rc | |||
|style="font-weight:bold" align="center" | Nonsense | |||
|style="font-weight:bold" align="center" | NA | |||
| align="center" | A3 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | BSA-Ab | |||
|style="font-weight:bold" align="center" valign="bottom" | p6/p4RC | |||
|style="font-weight:bold" align="center" valign="bottom" | Nonsense | |||
|style="font-weight:bold" align="center" valign="bottom" | NA | |||
| align="center" | A4 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample1A | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" | PA | |||
|style="font-weight:bold" align="center" | T1 | |||
| align="center" | A5 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample1B | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" | PA | |||
|style="font-weight:bold" align="center" | T1 | |||
| align="center" | A6 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample2A | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" | PA | |||
|style="font-weight:bold" align="center" | T2 | |||
| align="center" | A7 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample2B | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" valign="bottom" | PA | |||
|style="font-weight:bold" align="center" valign="bottom" | T2 | |||
| align="center" | A8 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample3A | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" | PA | |||
|style="font-weight:bold" align="center" | T3 | |||
| align="center" | H1 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample3B | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" valign="bottom" | PA | |||
|style="font-weight:bold" align="center" valign="bottom" | T3 | |||
| align="center" | H2 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample4A | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" | PA | |||
|style="font-weight:bold" align="center" | T4 | |||
| align="center" | H3 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample4B | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" | PA | |||
|style="font-weight:bold" align="center" | T4 | |||
| align="center" | H4 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | NTC | |||
|style="font-weight:bold" align="center" | p24/p4RC | |||
|style="font-weight:bold" align="center" | PA | |||
|style="font-weight:bold" align="center" | NA | |||
| align="center" | H5 | |||
| align="center" align="center" | 0 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | NTC | |||
|style="font-weight:bold" align="center" | p6/p12RC | |||
|style="font-weight:bold" align="center" | PB | |||
|style="font-weight:bold" align="center" | NA | |||
| align="center" | H6 | |||
| align="center" align="center" | 0 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | NTC | |||
|style="font-weight:bold" align="center" | p24/p12rc | |||
|style="font-weight:bold" align="center" | Nonsense | |||
|style="font-weight:bold" align="center" | NA | |||
| align="center" | H7 | |||
| align="center" align="center" | 0 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | NTC | |||
|style="font-weight:bold" align="center" valign="bottom" | p6/p4RC | |||
|style="font-weight:bold" align="center" valign="bottom" | Nonsense | |||
|style="font-weight:bold" align="center" | NA | |||
| align="center" | H8 | |||
| align="center" align="center" | 0 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 50 | |||
|} | |||
[[Category:ComboLock]] [[Category:20161103]] |
Latest revision as of 17:28, 7 November 2016
Antibody Binding Test (Started Yesterday)[edit]
Protocol[edit]
- qPCR
- Make master mix according to following recipe
- 233.1 uL nfH2O
- 277.5 uL SYBR Fast
- 11.1 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 120 uL TBE, 30 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 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
qPCR Sample Table[edit]
Sample | Condition | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | 2 ug BSA | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | 2 ug BSA | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No Antibody | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No Antibody | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | 20 ng BSA | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | 20 ng BSA | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | 200 pg BSA | H1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | 200 pg BSA | H2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR NTC | H3 | 25 | 0 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
- 20161104-qPCR-AntibodyTest.png
qPCR curve
- 2016-11-04-AntibodyTest-Gel1.png
Gel Image 1
- 2016-11-04-AntibodyTest-Gel2.png
Gel Image 2
Testing[edit]
Doing some QC control on the antibody experiment to figure out what went wrong. The first thing to test is the BSA Ab-oligo complexes.
- qPCR
- Make master mixes according to following recipes
- Add 47 uL master mix to each well
- Add 2 uL sample according to table in following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 160 uL TBE, 40 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 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Master Mix | No. Samples | Primers | uL Reverse Primer (10 uM) | uL Forward Primer (10 uM) | uL Kapa SYBR Fast | uL nfH2O |
PA | 10 | p24/p4RC | 10.1 | 10.1 | 252.5 | 212.1 |
PB | 2 | p6/p12RC | 2.1 | 2.1 | 52.5 | 44.1 |
Nonsense A | 2 | p24/p12rc | 2.1 | 2.1 | 52.5 | 44.1 |
Nonsense B | 2 | p6/p4RC | 2.1 | 2.1 | 52.5 | 44.1 |
Results[edit]
- 20161104-qPCR-QC-AntibodyTest-PosNeg.png
qPCR curve comparing positive controls (BSA) vs. negative controls (NTC)
- 20161104-qPCR-QC-AntibodyTest-TimeCourse.png
Time course comparison (negative controls included)
- 2016-11-04-AntibodyTest-QC-Gel1.png
Gel Image 1
- 2016-11-04-AntibodyTest-QC-Gel2.png
Gel Image 2
qPCR Sample Table[edit]
Sample | Primers | Primer Target | Time Point | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
BSA-Ab | p24/p4RC | PA | NA | A1 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA-Ab | p6/p12RC | PB | NA | A2 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA-Ab | p24/p12rc | Nonsense | NA | A3 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA-Ab | p6/p4RC | Nonsense | NA | A4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample1A | p24/p4RC | PA | T1 | A5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample1B | p24/p4RC | PA | T1 | A6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample2A | p24/p4RC | PA | T2 | A7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample2B | p24/p4RC | PA | T2 | A8 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample3A | p24/p4RC | PA | T3 | H1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample3B | p24/p4RC | PA | T3 | H2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample4A | p24/p4RC | PA | T4 | H3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample4B | p24/p4RC | PA | T4 | H4 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | p24/p4RC | PA | NA | H5 | 0 | 25 | 1 | 1 | 23 | 50 |
NTC | p6/p12RC | PB | NA | H6 | 0 | 25 | 1 | 1 | 23 | 50 |
NTC | p24/p12rc | Nonsense | NA | H7 | 0 | 25 | 1 | 1 | 23 | 50 |
NTC | p6/p4RC | Nonsense | NA | H8 | 0 | 25 | 1 | 1 | 23 | 50 |