Nov 22, 2019

Public workspaceDNA/RNA Radiolabeling Protocol

DOI
dx.doi.org/10.17504/protocols.io.8dshs6e
  • Liz O'Brien1,
  • Connor Tsuchida1
  • 1University of California, Berkeley
  • The Center for Genome Editing and Recording
Meredith Triplet
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DOI: dx.doi.org/10.17504/protocols.io.8dshs6e
Protocol Citation: Liz O'Brien, Connor Tsuchida 2019. DNA/RNA Radiolabeling Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8dshs6e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2019
Last Modified: November 22, 2019
Protocol Integer ID: 28818
Keywords: Radiolabeling, DNA, RNA, CasX, TS, NTS
Attachments
Icon for Radiolabeling_CasX_DNA_substrates.pdf
Radiolabeling_CasX_D...
141KB
Guidelines
CasX TS/NTS with non-hydrolysable spacers:

TS:

5’-CGCTAGCTACGT*T*T*G*A*T*T*T*T*C*T*G*C*T*G*C*A*G*G*A*TGAAATCCCGTAATCGCGC-3’

MW: 15664.2 g/mol
Concentration: X μM

*= phosphothioate, bold letters = PAM, italic letters = spacer

For 10 pmol of TS: X μl of stock


NTS:

5’-GCGCGATTACGGGATTTCAT*C*C*T*G*C*A*G*C*A*G*A*A*A*A*T*C*A*A*A*CGTAGCTAGCG-3’

MW: 15749.3 g/mol
Concentration: X μM

*= phosphothioate, bold letters = PAM, italic letters = spacer

For 10 pmol of NTS: X μl of substrate




Labelling reaction setup:

*TS:

XX ulDNA or RNA (10 pmoles)
2.5 ul10x PNK buffer
0.5 ulPNK enzyme
1.5 ulP32-gamma-ATP
XX mL dH2O (DEPC for labeling RNA) to 25 ul

*NTS:

XX ulDNA or RNA (10 pmoles)
2.5 ul10x PNK buffer
0.5 ulPNK enzyme
1.5 ulP32-gamma-ATP
XX mL dH2O (DEPC for labeling RNA) to 25 ul
Materials
MATERIALS
ReagentT4 Polynucleotide Kinase (3' phosphatase minus) - 200 unitsNew England BiolabsCatalog #M0236S
Reagent10X T4 PNK Reaction BufferNew England Biolabs
ReagentATP [γ-32P]- 3000Ci/mmol 10mCi/ml Lead 100 µCi (P32-gamma-ATP)Perkin ElmerCatalog #NEG002A100UC
ReagentHiTrap Desalting columns with Sephadex G-25 resinGE HealthcareCatalog #29048684


Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Set up labeling reaction:
X μlDNA or RNA (10 pmoles)
2.5 μl10x PNK buffer
0.5 μlPNK enzyme
1.5 μlP32-gamma-ATP
dH2O (DEPC for labeling RNA) to 25 μl

Note
Mix the DNA, buffer, enzyme, and H2O at the bench, and then add the DNA/enzyme mixture to ATP-filled tubes in a radioactive use area.

Incubate at Temperature37 °C for Duration00:30:00 .
Incubation
Heat inactivate the PNK at Temperature65 °C for Duration00:20:00 .
Prepare G25 columns (from GE, green box): vortex thoroughly, twist cap ¼ turn, snap off bottom, spin for Duration00:01:00 at Centrifigation3000 rpm to get rid of liquid.

Centrifigation
Add Amount50 µL H2O to a labeled eppendorf tube, place G25 column in it.
Pipetting
Add Amount25 µL H2O to each labeling reaction after heat inactivation is done.

Pipetting
Apply entire reaction (now 50 μl total) to G25 column resin.
Spin for Duration00:02:00 at Centrifigation3000 rpm .

Centrifigation
Since 50 μl H2O were in bottom of tube and you add your 50 μl reaction, you should end with up to 100 μl of Concentration100 nanomolar (nM) labeled DNA/RNA.

Measure Amount1 µL of each reaction with the black rad counter on shelf to get cpm readings.

Analyze