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

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'''Preparation'''
'''Preparation'''
* Work with samples on ice
* Work with samples on ice
-
* Ice RNase: H, SS III RT, and RNase out
+
* 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 2u of each sample plus  blank to the plate reader
+
* 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 8uL solutions of the lowest concentration for each cell line
+
*Need to make 8μL solutions of the lowest concentration for each cell line
-
# Sample 1: 7.1uL cells + 0.9 H2O = 2.5ug
+
# Sample 1: 7.1μL cells + 0.9 H2O = 2.5ug
-
# Sample 2: 8uL cells = 2.5ng
+
# Sample 2: 8μL cells = 2.5ng
-
# Sample 3: 8uL cells = 0.35ug
+
# Sample 3: 8μL cells = 0.35ug
-
# Sample 4: 2.2uL cells + H2O = 0.35ng
+
# 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 1uL of primer (Oligo dT)
+
* Add 1μL of primer (Oligo dT)
-
* Add 1u of dNTP
+
* 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 2uL RT Buffer, 4uL MgCl2, 2uL DTT, 1 uL RNaseOut, and 1uL SS III per rxn
+
* Make a MM for 4 rxns: need 2μL RT Buffer, 4μL MgCl2, 2μL DTT, 1μL RNaseOut, and 1μL SS III per rxn
-
* Add 10uL of MM to each rxn tube
+
* 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 1uL: of RNase H
+
* 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

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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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