Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2013/01/10

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(Autocreate 2013/01/10 Entry for Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2)
Current revision (21:52, 10 January 2013) (view source)
(→cDNA)
 
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==Entry title==
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==cDNA==
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* Insert content here...
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'''Preparation'''
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* Work with samples on ice
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* Ice: RNase H, SS III RT, and RNase out
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* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT
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'''Sample Reading'''
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* Add 2μL of each sample plus  blank to the plate reader
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# K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL)
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# K562 Sample 2: 0.385, 2.035, 308.3
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# SK-N-SH Sample 1: 0.054, 1.819, 43.3
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# SK-N-SH Sample 2: 0.201, 2.016, 160.7
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*Need to make 8μL solutions of the lowest concentration for each cell line
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# Sample 1: 7.1μL cells + 0.9 H2O = 2.5ug
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# Sample 2: 8μL cells = 2.5ng
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# Sample 3: 8μL cells = 0.35ug
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# Sample 4: 2.2μL cells + H2O = 0.35ng
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'''Part 1'''
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* Make sure all samples are filled up to 8uL (fill rest with water if not)
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* Add 1μL of primer (Oligo dT)
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* Add 1μL of dNTP
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* Incubate for 5 min @ 65°C
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* Ice for 1 min
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'''Part 2'''
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* Make a MM for 4 rxns: need 2μL RT Buffer, 4μL MgCl2, 2μL DTT, 1μL RNaseOut, and 1μL SS III per rxn
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* Add 10μL of MM to each rxn tube
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* Mix tubes gently, centrifuge for a few seconds
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'''Part 3'''
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* RT-PCR Machine, set up as follows:
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# Stage 1: 50 min @ 50°C
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# Stage 2: 5 min @ 85°C
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# Stage 3: Infinity @ 4°C
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* Add 1μL: of RNase H
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* Incubate @ 37°C for 20 min
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* Store at -20°C
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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 2μL of each sample plus blank to the plate reader
  1. K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL)
  2. K562 Sample 2: 0.385, 2.035, 308.3
  3. SK-N-SH Sample 1: 0.054, 1.819, 43.3
  4. SK-N-SH Sample 2: 0.201, 2.016, 160.7
  • Need to make 8μL solutions of the lowest concentration for each cell line
  1. Sample 1: 7.1μL cells + 0.9 H2O = 2.5ug
  2. Sample 2: 8μL cells = 2.5ng
  3. Sample 3: 8μL cells = 0.35ug
  4. Sample 4: 2.2μL cells + H2O = 0.35ng

Part 1

  • Make sure all samples are filled up to 8uL (fill rest with water if not)
  • Add 1μL of primer (Oligo dT)
  • Add 1μL of dNTP
  • Incubate for 5 min @ 65°C
  • Ice for 1 min

Part 2

  • Make a MM for 4 rxns: need 2μL RT Buffer, 4μL MgCl2, 2μL DTT, 1μL RNaseOut, and 1μL SS III per rxn
  • Add 10μL of MM to each rxn tube
  • Mix tubes gently, centrifuge for a few seconds

Part 3

  • RT-PCR Machine, set up as follows:
  1. Stage 1: 50 min @ 50°C
  2. Stage 2: 5 min @ 85°C
  3. Stage 3: Infinity @ 4°C
  • Add 1μL: of RNase H
  • Incubate @ 37°C for 20 min
  • Store at -20°C
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