Daniel:Notebook/ComboLock/2016-9-29: Difference between revisions
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | C4-C2 Amplicon (Final) | | height="15" valign="bottom" | C4-C2 Amplicon (Final) | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 78.3 | ||
|} | |} | ||
Line 49: | Line 49: | ||
<ol type="A"> | <ol type="A"> | ||
<li>Follow reaction table below to set up 4 reactions</li> | <li>Follow reaction table below to set up 4 reactions</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" | |||
| width="105" height="45" | Sample | |||
| width="65" | 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 primer12RC | |||
| width="65" | 10 uM AmpR6.3-IndXX | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1A | |||
| align="center" align="center" valign="bottom" | 1.00E+09 | |||
| align="center" valign="bottom" | A1 | |||
| align="center" align="center" valign="bottom" | 37 | |||
| 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 1B | |||
| align="center" align="center" valign="bottom" | 1.00E+09 | |||
| align="center" valign="bottom" | A2 | |||
| align="center" align="center" valign="bottom" | 37 | |||
| 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:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1C | |||
| align="center" align="center" valign="bottom" | 1.00E+09 | |||
| align="center" valign="bottom" | A3 | |||
| align="center" align="center" valign="bottom" | 37 | |||
| 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" | NTC | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" valign="bottom" | A4 | |||
| align="center" align="center" valign="bottom" | 37 | |||
| 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>Make the following master mix</li> | <li>Make the following master mix</li> | ||
<ol type="a"> | <ol type="a"> | ||
Line 72: | Line 137: | ||
<gallery perrow=2 widths=300px heights=300px> | <gallery perrow=2 widths=300px heights=300px> | ||
File:| | File:20160929-qPCRAmpFTest.png|qPCR Curve | ||
File:| | File:2016-09-29-qPCR-Primer12RC.png|Gel Image | ||
</gallery> | </gallery> | ||
[[Category:ComboLock]] [[Category:20160928]] | ===Discussion=== | ||
As I expected there is no extra product (or very little) being formed. | |||
[[Category:ComboLock]] [[Category:20160928]] [[Category:20160929]] |
Latest revision as of 20:25, 30 September 2016
Positive Control Amplicon Production (PCAmp4; Started yesterday)[edit]
Protocol[edit]
- Size Selection
- Centrifuge at 12000 rpm for 1.5 minutes
- Extract the supernatant and place into NanoSep column
- Centrifuge at 12000 rpm for 1.5 minutes
- Ethanol Precipitation
- Move sample to a fresh 2 mL tube
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue
- Store at for 6 hours
- Pellet by centrifuging for 25 minutes at 12000rpm at 4C
- Remove supernatant and add 750 uL chilled 70% EtOH
- Centrifuge for 12 minutes at 12000rpm at 4C
- Remove supernatant and spin in vacuum centrifuge for 10 minutes
- Add 50 uL nfH2O and resuspend every tube in series;
- Measure final concentration in nanodrop
Results[edit]
Sample | ng/uL ssDNA |
C4-C2 Amplicon (Final) | 78.3 |
AmpF Test[edit]
Since I'm testing whether the AmpF primer (which has LinkerR that matches to primer2 and therefore has a potential off-target binding) creates the wrong product or not. To test this I'll be using the PCR primer Primer12-RC, which should be a forward primer for amplifying off the circular target. In conjuction with AmpR6.3, this should produce a 157bp product. Let's see what happens.
Protocol[edit]
- qPCR
- Follow reaction table below to set up 4 reactions
- Make the following master mix
- 71.75 uL nfH2O
- 2.05 uL Primer12RC
- 20.5 uL AmpR6.3-34
- 102.5 uL SYBR Fast
- Aliquot 48 uL master mix into each well
- Add 2 uL sample to each 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
- 20160929-qPCRAmpFTest.png
qPCR Curve
- 2016-09-29-qPCR-Primer12RC.png
Gel Image
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM primer12RC | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1A | 1.00E+09 | A1 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 1.00E+09 | A2 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 1.00E+09 | A3 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | A4 | 37 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Results[edit]
Discussion[edit]
As I expected there is no extra product (or very little) being formed.