Daniel:Notebook/ComboLock/2016-7-28: Difference between revisions

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<li>Prepare 20 uL KLN mix</li>
<li>Prepare 20 uL KLN mix</li>
<li>Prepare  
<li>Prepare 4.1x master mix of Phusion mix</li>
<li>Add 2 uL KLN mix to 20 uL of reaction</li>
<li>Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5</li>
<li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li>
<li>Incubate at 55C overnight (XX hours) </li>
<li>Incubate at 55C overnight (XX hours) </li>
<li>Continued [[Daniel:Notebook/ComboLock/2016-7-29|tomorrow]]</li>
<li>Continued [[Daniel:Notebook/ComboLock/2016-7-29|tomorrow]]</li>

Latest revision as of 20:41, 28 July 2016

Phusion Test[edit]

Back to Calendar

Hemo Klentaq has strand displacement activity, which could be a potential (big) problem for latch-padlock circularization. Therefore, on Chris and Andrew's recommendation I will try a test using Phusion instead of Hemo Klentaq.

Buffers[edit]

KLN Mix

Reagent Stock uL added
Amp Ligase Buffer 10X 2
dNTPs 100 uM 2
Hemo Klentaq NA 4
Amp Ligase 5 U/uL 2
nfH2O NA 10

Phusion Buffer

Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (4.1x) (uL)
NAD+ 5 mM 40 nmol 8 32.8
dNTP 1 mM 600 pmol 0.6 2.46
Betaine 5 M 15 umol 3 12.3
10X AmpLigase Buffer 10X 1X 2 8.2
Amp Ligase 5 U/uL 10 U 2 8.2
Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 13.12
nf H2O     1.2 4.92
Total     20 82

Protocol[edit]

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter
      1. Human - 3.05E5 cells/mL; Mouse - 9.75E5 cells/mL (from 7-25-2016)
    2. Add cells to a new tube such that there are 30000 cells present per sample
      1. Human- 350 uL (3 samples) uL; Mouse- 30 uL
    3. Pellet cells via centrifugation at 600g for 3 min
    4. Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    5. Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
    6. Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    7. Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
    8. Pellet cells via centrifugation at 600g for 3 min
    9. Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O
    10. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    11. Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample
    12.   C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) U87MG 3T3 Cells Treatment
      Sample 1 X X X   Klentaq
      Sample 2 X X X   Phusion
      Sample 3     X   Phusion
      Sample 4 X X   X Phusion
      Sample 5 X X     Phusion
    13. Incubate probes at 40C for 1 hour with vigorous agitation
    14. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    15. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
  2. Latch and Padlock Hybridization
    1. Pellet at 600xg for 3 min and remove supernatant
    2. Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0001-68
    3. Incubate for 30 min at 37C
    4. Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
    5. Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
    6. Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
      1. For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
    7. Incubate for 30 min at 37C
    8. Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
    9. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    10. Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
  3. Circularization
    1. Prepare 20 uL KLN mix
    2. Prepare 4.1x master mix of Phusion mix
    3. Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
    4. Add 2 uL KLN mix to 20 uL of reaction for sample 1
    5. Incubate at 55C overnight (XX hours)
    6. Continued tomorrow