Daniel:Notebook/ComboLock/2016-9-22: Difference between revisions
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*Primer concentration may affect the generation of all the products, but especially the primer dimers | *Primer concentration may affect the generation of all the products, but especially the primer dimers | ||
For reference see the | For reference see the [[Daniel:Notebook/ComboLock/2016-9-5|size select gel from 9-5-16]] which shows the three off target products that occur. | ||
==Protocol== | ==Protocol== |
Revision as of 18:51, 22 September 2016
PCR Testing
I'm going to do some testing with the PCR conditions to see if I can remove the off-target products. Right now I'm testing against several potential sources:
- Extension time may have something to do with the higher MW products (500+bp)
- Primer annealing temp may have something to do with the primer-dimer products (add analysis link)
- Primer concentration may affect the generation of all the products, but especially the primer dimers
For reference see the size select gel from 9-5-16 which shows the three off target products that occur.
Protocol
Tube No (A-C) | Dilution | Molecules Available |
0 | 1 | 1E11 |
1 | 10 | 1E10 |
- 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