Revision as of 17:40, 7 November 2016 by >Djacobse
Cell Test with RCA
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This experiment is designed to see if I can get mRNA targeting C-Probes to work in cells, this time with RCA. I'll be trying both VIM and SOD1, just to have two separate genes.
Protocol
Pellet cells via centrifugation at 600g for 3 min
Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
Pellet cells via centrifugation at 600g for 3 min
Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O
Heat C probes to 90C for 5 min; Chill probes on ice to quench
Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample
Incubate probes at 40C for 1 hour with vigorous agitation
Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0201-68 and 100 nM Latch0001
- Incubate for 30 min at 37C
- Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
- Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
Circularization
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction for sample 1
- Incubate at 55C overnight (XX hours)
- Continued tomorrow