Matt:LabNotes/2016-3-31: Difference between revisions
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==Link PKP2_control oligo to Streptavidin Bead== | ==Link PKP2_control oligo to Streptavidin Bead== | ||
#Make 2X B&W (Binding and Wash) Buffer | |||
#*16ml 5uM NaCl + 80ul 5uM EDTA + 100ul 4uM Tris-HCl + 23.82ml H2O | |||
#Resuspend beads by vortexing | |||
#Transfer 100ul of beads (10ug/ul) to new tube | |||
#Pull down by magnet 2min and remove supernatant | |||
#Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant | |||
#Resuspend in 200ul 2X B&W Buffer | |||
#Add 200ul of 2.5uM PKP2_Control | |||
#Incubate 15min at RT gently rotating | |||
#Pull down with magnet | |||
#Wash 3x with 1X B&W Buffer | |||
#Resuspend in 1ml 1X PBS | |||
==Measure DNA Concentration== | ==Measure DNA Concentration== |
Revision as of 19:30, 8 April 2016
Bead with PKP2 Target for DARTFISH Positive Control=
- Using PA gel may prevent diffusion of probes and enzymes during DARTFISH
- To test this and to have a positive control for future experiments need to design a synthetic target
- Attach target to magnetic streptavidin bead
- Design oligonucleotide with biotin 5' modification
- Chose PKP2 gene because haven't detected it in any DARTFISH samples
- Also this specific padlock probe has very high efficiency when measured in tube
PKP2_control /5BiosG/AAAAAAGAGATGGCTGTCTTTTTCACACTTGGGTCACCAACATGCAGCATCTTTC
Link PKP2_control oligo to Streptavidin Bead
- Make 2X B&W (Binding and Wash) Buffer
- 16ml 5uM NaCl + 80ul 5uM EDTA + 100ul 4uM Tris-HCl + 23.82ml H2O
- Resuspend beads by vortexing
- Transfer 100ul of beads (10ug/ul) to new tube
- Pull down by magnet 2min and remove supernatant
- Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
- Resuspend in 200ul 2X B&W Buffer
- Add 200ul of 2.5uM PKP2_Control
- Incubate 15min at RT gently rotating
- Pull down with magnet
- Wash 3x with 1X B&W Buffer
- Resuspend in 1ml 1X PBS