Daniel:Notebook/ComboLock/2016-9-30: Difference between revisions
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| height="15" valign="bottom" | Amp Ligase Buffer | | height="15" valign="bottom" | Amp Ligase Buffer | ||
| align="center" valign="bottom" | 10X | | align="center" valign="bottom" | 10X | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 6 | ||
|- style="background-color:#D9D9D9;font-size:12pt" | |- style="background-color:#D9D9D9;font-size:12pt" | ||
| height="15" valign="bottom" | dNTPs | | height="15" valign="bottom" | dNTPs | ||
| align="center" valign="bottom" | 100 uM | | align="center" valign="bottom" | 100 uM | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 6 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Hemo Klentaq | | height="15" valign="bottom" | Hemo Klentaq | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 12 | ||
|- style="background-color:#D9D9D9;font-size:12pt" | |- style="background-color:#D9D9D9;font-size:12pt" | ||
| height="15" valign="bottom" | Amp Ligase | | height="15" valign="bottom" | Amp Ligase | ||
| align="center" valign="bottom" | 5 U/uL | | align="center" valign="bottom" | 5 U/uL | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 6 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 30 | ||
|} | |} |
Revision as of 22:03, 30 September 2016
AmpR Test
Yesterday I tested the AmpR primer (the names are actually confusing, the name is the primer I'm NOT using. So yesterday I amplified off the circularized product using AmpR (which binds to the LinkerL region of the circularized product) and primer12RC, which binds to the amplified product. Now I'm going to test the amplification of using the AmpF primer. If AmpF is the culprit (which is what I expect) then I should see off-target binding. If the off-target products require both AmpF and AmpR, I shouldn't see off-target product.
Protocol
- qPCR
- Follow reaction table below to set up 4 reactions
- Make the following master mix
- 90.2 uL nfH2O
- 2.05 uL Primer2
- 2.05 uL AmpF6.4Sol
- 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
- 20160930-qPCRCurve-Primer2.png
qPCR curve
- 2016-09-30-qPCR-Primer2.png
Gel image
- Latch and Padlock Hybridization
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL oligo per sample to bead solution; incubate at RT for 5 min
- Apply magnet for 30 sec and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube
- Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C with intermittent mixing
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 30 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight; continued tomorrow
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF6.4Sol | 100 uM primer2 | H2O | Total Volume (uL) |
Sample 0A | 1.00E+10 | A1 | NA | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 0B | 1.00E+10 | A2 | NA | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 0C | 1.00E+10 | A3 | NA | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
NTC | 0 | A4 | NA | 0 | 25 | 0.5 | 0.5 | 24 | 50 |
Results
Discussion
Well, this pretty much confirms it. AmpF is the problem and is producing off-target products. Note that the expected product is the 168 bp band, but a primer dimer caused by the off-target binding would produce a product of approximately 75bp. We see a clear band at 75 bp, meaning that the AmpF-primer2 binding is likely too strong. Hopefully with changing primer2 to primer24 (see PCAmp4 production) for further details.
Positive Control Amplicon 4 Test
PCAmp4 uses the primer24 adapter instead of primer2 and should not have the same problems in amplification that the primer2-based system has. See Wednesday for the production of PCAmp4 (C4-C2).
Dilutions
Tube No | Dilution | Molecules Available |
0 | 1 | 100000000000 |
1 | 10 | 10000000000 |
2 | 100 | 1.E+09 |
3 | 1000 | 1.E+08 |
4 | 10000 | 1.E+07 |
5 | 100000 | 1.E+06 |
Buffers
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 6 |
dNTPs | 100 uM | 6 |
Hemo Klentaq | NA | 12 |
Amp Ligase | 5 U/uL | 6 |
nfH2O | NA | 30 |