Daniel:Notebook/ComboLock/2016-10-12: Difference between revisions
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<li>Make the following master mix</li> | <li>Make the following master mix</li> | ||
<ol type="a"> | <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> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
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|} | |} | ||
<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> | |||
[[Category:ComboLock]] [[Category:20161007]] | [[Category:ComboLock]] [[Category:20161007]] |
Revision as of 22:36, 12 October 2016
Padlock0301 Test (Started Friday 10-7)
Testing the amplification using the forward (AmpF-CL-v2) and primer24 reverse set, as well as some dilutions.
Protocol
- 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
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
- 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
- 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
- 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
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 |