Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2013/01/10: Difference between revisions
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'''Preparation''' | '''Preparation''' | ||
* Work with samples on ice | * Work with samples on ice | ||
* Ice RNase | * Ice: RNase H, SS III RT, and RNase out | ||
* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT | * Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT | ||
'''Sample Reading''' | '''Sample Reading''' | ||
* Add | * Add 2μL of each sample plus blank to the plate reader | ||
# K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL) | # K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL) | ||
# K562 Sample 2: 0.385, 2.035, 308.3 | # K562 Sample 2: 0.385, 2.035, 308.3 | ||
# SK-N-SH Sample 1: 0.054, 1.819, 43.3 | # SK-N-SH Sample 1: 0.054, 1.819, 43.3 | ||
# SK-N-SH Sample 2: 0.201, 2.016, 160.7 | # SK-N-SH Sample 2: 0.201, 2.016, 160.7 | ||
*Need to make | *Need to make 8μL solutions of the lowest concentration for each cell line | ||
# Sample 1: 7. | # Sample 1: 7.1μL cells + 0.9 H2O = 2.5ug | ||
# Sample 2: | # Sample 2: 8μL cells = 2.5ng | ||
# Sample 3: | # Sample 3: 8μL cells = 0.35ug | ||
# Sample 4: 2. | # Sample 4: 2.2μL cells + H2O = 0.35ng | ||
'''Part 1''' | '''Part 1''' | ||
* Make sure all samples are filled up to 8uL (fill rest with water if not) | * Make sure all samples are filled up to 8uL (fill rest with water if not) | ||
* Add | * Add 1μL of primer (Oligo dT) | ||
* Add | * Add 1μL of dNTP | ||
* Incubate for 5 min @ 65°C | * Incubate for 5 min @ 65°C | ||
* Ice for 1 min | * Ice for 1 min | ||
'''Part 2''' | '''Part 2''' | ||
* Make a MM for 4 rxns: need | * Make a MM for 4 rxns: need 2μL RT Buffer, 4μL MgCl2, 2μL DTT, 1μL RNaseOut, and 1μL SS III per rxn | ||
* Add | * Add 10μL of MM to each rxn tube | ||
* Mix tubes gently, centrifuge for a few seconds | * Mix tubes gently, centrifuge for a few seconds | ||
'''Part 3''' | '''Part 3''' | ||
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# Stage 2: 5 min @ 85°C | # Stage 2: 5 min @ 85°C | ||
# Stage 3: Infinity @ 4°C | # Stage 3: Infinity @ 4°C | ||
* Add | * Add 1μL: of RNase H | ||
* Incubate @ 37°C for 20 min | * Incubate @ 37°C for 20 min | ||
* Store at -20°C | * Store at -20°C |
Revision as of 18:52, 10 January 2013
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cDNAPreparation
Sample Reading
Part 1
Part 2
Part 3
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