Daniel:Notebook/ComboLock/2016-8-29: Difference between revisions
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<li>qPCR X2-Primers</li> | <li>qPCR X2-Primers</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC | <li>Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC</li> | ||
<li>Load lanes according to table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B7DEE8;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="31" | Sample Name | |||
| width="65" | Sample No. | |||
| width="65" | qPCR Lane | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | Pure C1-C2 | |||
| Sample 00 | |||
| A1 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 1E+11 | |||
| Sample 0 | |||
| B1 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 1E+10 | |||
| Sample 1 | |||
| C1 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 1E+09 | |||
| Sample 2 | |||
| D1 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 1E+08 | |||
| Sample 3 | |||
| E1 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 1E+07 | |||
| Sample 4 | |||
| F1 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 1E+06 | |||
| Sample 5 | |||
| G1 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 1E+05 | |||
| Sample 6 | |||
| H1 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 10000 | |||
| Sample 7 | |||
| A12 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 1000 | |||
| Sample 8 | |||
| B12 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 100 | |||
| Sample 9 | |||
| C12 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 10 | |||
| Sample 10 | |||
| D12 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 1 | |||
| Sample 11 | |||
| E12 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| height="15" | NTC | |||
| NTC | |||
| F12 | |||
|} | |||
<li>Master Mix</li> | <li>Master Mix</li> | ||
<ol type="a"> | <ol type="a"> | ||
Line 28: | Line 107: | ||
<li>72C 2 min</li> | <li>72C 2 min</li> | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol></ol> | </ol></ol> | ||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix together 72 uL TBE and 18 uL 6X loading dye in a 0.2mL tube</li> | |||
<li>Add 1 or 2 uL sample according to table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#E6B8B7;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="31" | Gel Lane | |||
| width="86" | Sample Name | |||
| width="65" | qPCR Lane | |||
| width="65" | uL Sample | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 2 | |||
| 25 bp Ladder | |||
| align="center" | | |||
| align="center" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 3 | |||
| 1E11 Molecule | |||
| B1 | |||
| align="center" | 1 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 4 | |||
| 1E9 Molecule | |||
| D1 | |||
| align="center" | 1 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 5 | |||
| 1E7Molecule | |||
| F1 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 6 | |||
| 1E5 Molecule | |||
| H1 | |||
| align="center" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 7 | |||
| 1000 Molecule | |||
| B12 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 8 | |||
| 10 Molecule | |||
| D12 | |||
| align="center" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
| align="center" height="15" | 9 | |||
| 1 Molecule | |||
| D13 | |||
| align="center" | 2 | |||
|} | |||
<li>Add 10 uL mix to well</li> | |||
<li>Run gel for 25 minutes at 250V</li> | |||
<li>Stain for 3 min with 3 uL SYBR gold</li> | |||
<li>Image in gel doc</li> | |||
</ol></ol> | |||
===Results=== | ===Results=== | ||
[[Image:20160829-X2Dilution-Primers-qPCR.png|600px]] | [[Image:20160829-X2Dilution-Primers-qPCR.png|600px]] |
Revision as of 17:33, 29 August 2016
Positive Control Dilution Test (Started Friday)
Protocol
Today I'm testing the samples using the P2 and P12RC amplification primers. Yesterday included the original amplicon materials; this qPCR should be only the DNA still attached to beads.
- qPCR X2-Primers
- Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
- Load lanes according to table
- Master Mix
- 352.5 uL KAPA SyberFast MM
- 310.2 uL nfH2O
- 7.05 uL primer2
- 7.05 uL primer12RC
- Add 48 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x32
- 72C 2 min
- 16C hold
- TBE Gel
- Mix together 72 uL TBE and 18 uL 6X loading dye in a 0.2mL tube
- Add 1 or 2 uL sample according to table
- Add 10 uL mix to well
- Run gel for 25 minutes at 250V
- Stain for 3 min with 3 uL SYBR gold
- Image in gel doc
Sample Name | Sample No. | qPCR Lane |
Pure C1-C2 | Sample 00 | A1 |
1E+11 | Sample 0 | B1 |
1E+10 | Sample 1 | C1 |
1E+09 | Sample 2 | D1 |
1E+08 | Sample 3 | E1 |
1E+07 | Sample 4 | F1 |
1E+06 | Sample 5 | G1 |
1E+05 | Sample 6 | H1 |
10000 | Sample 7 | A12 |
1000 | Sample 8 | B12 |
100 | Sample 9 | C12 |
10 | Sample 10 | D12 |
1 | Sample 11 | E12 |
NTC | NTC | F12 |
Gel Lane | Sample Name | qPCR Lane | uL Sample |
2 | 25 bp Ladder | 2 | |
3 | 1E11 Molecule | B1 | 1 |
4 | 1E9 Molecule | D1 | 1 |
5 | 1E7Molecule | F1 | 2 |
6 | 1E5 Molecule | H1 | 2 |
7 | 1000 Molecule | B12 | 2 |
8 | 10 Molecule | D12 | 2 |
9 | 1 Molecule | D13 | 2 |