Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2013/01/10: Difference between revisions
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Caroline Hom (talk | contribs) (Autocreate 2013/01/10 Entry for Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2) |
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== | ==cDNA== | ||
* | '''Preparation''' | ||
* Work with samples on ice | |||
* Ice RNase: H, SS III RT, and RNase out | |||
* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT | |||
'''Sample Reading''' | |||
* Add 2u 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 2: 0.385, 2.035, 308.3 | |||
# SK-N-SH Sample 1: 0.054, 1.819, 43.3 | |||
# SK-N-SH Sample 2: 0.201, 2.016, 160.7 | |||
*Need to make 8uL solutions of the lowest concentration for each cell line | |||
# Sample 1: 7.1uL cells + 0.9 H2O = 2.5ug | |||
# Sample 2: 8uL cells = 2.5ng | |||
# Sample 3: 8uL cells = 0.35ug | |||
# Sample 4: 2.2uL cells + H2O = 0.35ng | |||
'''Part 1''' | |||
* Make sure all samples are filled up to 8uL (fill rest with water if not) | |||
* Add 1uL of primer (Oligo dT) | |||
* Add 1u of dNTP | |||
* Incubate for 5 min @ 65°C | |||
* Ice for 1 min | |||
'''Part 2''' | |||
* Make a MM for 4 rxns: need 2uL RT Buffer, 4uL MgCl2, 2uL DTT, 1 uL RNaseOut, and 1uL SS III per rxn | |||
* Add 10uL of MM to each rxn tube | |||
* Mix tubes gently, centrifuge for a few seconds | |||
'''Part 3''' | |||
* RT-PCR Machine, set up as follows: | |||
# Stage 1: 50 min @ 50°C | |||
# Stage 2: 5 min @ 85°C | |||
# Stage 3: Infinity @ 4°C | |||
* Add 1uL: of RNase H | |||
* Incubate @ 37°C for 20 min | |||
* Store at -20°C | |||
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Revision as of 18:48, 10 January 2013
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cDNAPreparation
Sample Reading
Part 1
Part 2
Part 3
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