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>Djacobse (Created page with "=Padlock0301 Test (Started Friday 10-7)= Back to Calendar Testing the amplification using the forward (...") |
>Djacobse |
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==Protocol== | ==Protocol== | ||
<ol> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make the following master mixes</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#95B3D7;font-size:12pt;font-weight:bold" align="center" | |||
| width="105" height="30" | Master Mix | |||
| width="65" | nfH20 | |||
| width="65" | AmpF-CL-v2 | |||
| width="105" | Reverse Primer | |||
| width="104" | Rev. Primer Conc. (uM) | |||
| width="83" | Amt. Reverse Primer | |||
| width="105" | Kapa SYBR Fast MM | |||
| width="65" | Total | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | MM1 | |||
| align="center" | 90.2 | |||
| align="center" | 2.05 | |||
| primer24 | |||
| align="center" | 100 | |||
| align="center" | 2.05 | |||
| align="center" | 102.5 | |||
| align="center" | 196.8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" | |||
| height="15" | MM2 | |||
| align="center" | 93.48 | |||
| align="center" | 0.41 | |||
| AmpR6.3-27 | |||
| align="center" | 100 | |||
| align="center" | 0.41 | |||
| align="center" | 102.5 | |||
| align="center" | 196.8 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | MM3 | |||
| align="center" | 90.2 | |||
| align="center" | 2.05 | |||
| AmpR6.3-28 | |||
| align="center" | 10 | |||
| align="center" | 2.05 | |||
| align="center" | 102.5 | |||
| align="center" | 196.8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" | |||
| height="15" | MM4 | |||
| align="center" | 93.48 | |||
| align="center" | 0.41 | |||
| AmpR6.3-29 | |||
| align="center" | 10 | |||
| align="center" | 0.41 | |||
| align="center" | 102.5 | |||
| align="center" | 196.8 | |||
|} | |||
<li>Set up qPCR reactions according to table in next section</li> | |||
<li>Aliquot 48 uL correct master mix into each well</li> | |||
<li>Add 2 uL sample to appropriate well</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 x26</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 48 uL TBE, 12 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> | |||
===qPCR Table=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
Line 247: | Line 339: | ||
|} | |} | ||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:2016-10-12-qPCR-Padlock0301-PrimerTest.png|qPCR | |||
File:2016-10-12-Padlock0301-PrimerTest-Gel1.png|Gel Image 1 | |||
File:2016-10-12-Padlock0301-PrimerTest-Gel2.png|Gel Image 2 | |||
</gallery> | |||
==Library Prep== | |||
<ol> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make the following master mix</li> | |||
<ol type="a"> | |||
<li>71.75 uL nfH2O</li> | |||
<li>2.05 uL AmpF-CL-6.4</li> | |||
<li>102.5 uL Kapa SYBR Fast</li></ol> | |||
<li>Aliquot 43 uL master mix into each well</li> | |||
<li>Prep samples according to following table</li> | |||
{| 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="74" | Template Molecule Count | |||
| width="65" | Lane | |||
| width="65" | AmpR Index | |||
| width="65" | Sample Vol (uL) | |||
| width="65" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 100 uM AmpF-CL-v2 | |||
| width="65" | 10 uM AmpR6.3-IndXX | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1A | |||
| align="center" valign="bottom" | 10 pmol | |||
| align="center" valign="bottom" | A1 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1B | |||
| align="center" valign="bottom" | 10 pmol | |||
| align="center" valign="bottom" | B1 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1C | |||
| align="center" valign="bottom" | 10 pmol | |||
| align="center" valign="bottom" | C1 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | NTC | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" valign="bottom" | D1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 19.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|} | |||
<li>Add 2 uL sample to appropriate well</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 x26</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
[[Image:20161012-qpCR-Padlock0301-LibraryPrep.png|500px]] | |||
<li>Size Select Gel</li> | |||
<ol type="A"> | |||
<li>Mix 20 uL sample and 4 uL 6x loading dye into separate tubes for each sample</li> | |||
<li>Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube</li> | |||
<li>Aliquot 24 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> | |||
<li>Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li> | |||
<li>Image in gel doc</li> | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:2016-10-12-Padlock0301-SizeSelect.png|Before image | |||
File:2016-10-12-Padlock0301-SizeSelect-After.png|After image | |||
</gallery> | |||
<li>Centrifuge at 12000 rpm for 1.5 minutes</li> | |||
<li>Add 500 uL TE to each sample</li> | |||
<li>Incubate overnight with vigorous shaking; continued [[Daniel:Notebook/ComboLock/2016-10-13|tomorrow]]</li> | |||
</ol></ol> | |||
[[Category:ComboLock]] [[Category:20161007]] |
Latest revision as of 17:08, 13 October 2016
Padlock0301 Test (Started Friday 10-7)[edit]
Testing the amplification using the forward (AmpF-CL-v2) and primer24 reverse set, as well as some dilutions.
Protocol[edit]
- qPCR
- Make the following master mixes
- Set up qPCR reactions according to table in next section
- Aliquot 48 uL correct master mix into each well
- Add 2 uL sample to appropriate well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 48 uL TBE, 12 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 | nfH20 | AmpF-CL-v2 | Reverse Primer | Rev. Primer Conc. (uM) | Amt. Reverse Primer | Kapa SYBR Fast MM | Total |
MM1 | 90.2 | 2.05 | primer24 | 100 | 2.05 | 102.5 | 196.8 |
MM2 | 93.48 | 0.41 | AmpR6.3-27 | 100 | 0.41 | 102.5 | 196.8 |
MM3 | 90.2 | 2.05 | AmpR6.3-28 | 10 | 2.05 | 102.5 | 196.8 |
MM4 | 93.48 | 0.41 | AmpR6.3-29 | 10 | 0.41 | 102.5 | 196.8 |
qPCR Table[edit]
Sample | Molecule Count | Lane | Reverse Primer | Reverse Primer Conc. (uM) | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | Reverse Primer | 100 uM AmpF | H2O | Total Volume (uL) | Master Mix |
Sample 0A | 10 pmol | A1 | primer24 | 100 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM1 |
Sample 0B | 10 pmol | A2 | primer24 | 100 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM1 |
Sample 0C | 10 pmol | A3 | primer24 | 100 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM1 |
NTC (rev) | 0 | A4 | primer24 | 100 | 0 | 25 | 0.5 | 0.5 | 24 | 50 | MM1 |
Sample 0A | 10 pmol | A1 | AmpR6.3-27 | 100 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 0B | 10 pmol | A2 | AmpR6.3-27 | 100 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 0C | 10 pmol | A3 | AmpR6.3-27 | 100 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
NTC (rev) | 0 | A4 | AmpR6.3-27 | 100 | 0 | 25 | 0.1 | 0.1 | 24 | 50 | MM2 |
Sample 0A | 10 pmol | A1 | AmpR6.3-28 | 10 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 0B | 10 pmol | A2 | AmpR6.3-28 | 10 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 0C | 10 pmol | A3 | AmpR6.3-28 | 10 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
NTC (rev) | 0 | A4 | AmpR6.3-28 | 10 | 0 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 0A | 10 pmol | A1 | AmpR6.3-29 | 10 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
Sample 0B | 10 pmol | A2 | AmpR6.3-29 | 10 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
Sample 0C | 10 pmol | A3 | AmpR6.3-29 | 10 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
NTC (rev) | 0 | A4 | AmpR6.3-29 | 10 | 0 | 25 | 0.1 | 0.1 | 24 | 50 | MM4 |
Results[edit]
- 2016-10-12-qPCR-Padlock0301-PrimerTest.png
qPCR
- 2016-10-12-Padlock0301-PrimerTest-Gel1.png
Gel Image 1
- 2016-10-12-Padlock0301-PrimerTest-Gel2.png
Gel Image 2
Library Prep[edit]
- qPCR
- Make the following master mix
- 71.75 uL nfH2O
- 2.05 uL AmpF-CL-6.4
- 102.5 uL Kapa SYBR Fast
- Aliquot 43 uL master mix into each well
- Prep samples according to following table
- Add 2 uL sample to appropriate well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
- Size Select Gel
- Mix 20 uL sample and 4 uL 6x loading dye into separate tubes for each sample
- Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
- Aliquot 24 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
- Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- Image in gel doc
- 2016-10-12-Padlock0301-SizeSelect.png
Before image
- 2016-10-12-Padlock0301-SizeSelect-After.png
After image
- Centrifuge at 12000 rpm for 1.5 minutes
- Add 500 uL TE to each sample
- Incubate overnight with vigorous shaking; continued tomorrow
Sample | Template Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF-CL-v2 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1A | 10 pmol | A1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 10 pmol | B1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 10 pmol | C1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | D1 | 25 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
File:20161012-qpCR-Padlock0301-LibraryPrep.png