Daniel:Notebook/ComboLock/2016-10-12: Difference between revisions
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==Library Prep== | ==Library Prep== | ||
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<li>qPCR</li> | |||
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<li>Make the following master mix</li> | |||
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{| 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> | ||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="105" height="45" | Sample | ||
| width="74" | Template Molecule Count | | width="74" | Template Molecule Count | ||
| width="65" | Lane | | width="65" | Lane |
Revision as of 22:33, 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
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 |