AMPK Methods and Protocols

4. Complete EMEM culture medium: Eagle’s Minimum Essen-
   tial Medium (EMEM), 20% (v/v) FBS, nonessential amino
   acids, 100 units/ml penicillin, and 100μg/ml streptomycin.


5. Trypsin/EDTA solution: 0.25% (w/v) Trypsin,
   0.53 mM EDTA.


6. 50μm cell strainer.


7. Cell sorting buffer: PBS (without Ca2+/Mg2+), 25 mM
   HEPES, pH 7, 1% (v/v) FBS, 1 mM EDTA.


8. 12 ml FACS tube.


9. Flow cytometer.

2.4 Screening 1. GeneScan™500ROX.

2. PCR Primers:
   
   
   • PRKAA1 primers:forward 5^0 -FAM-ATCACCAGGATCC
     TTTGGCA-3^0 and reverse 5^0 -TGCTTTCCTTACACCTT
     GGTG-3^0.
   
   
   • PRKAA2 primers: forward 5^0 -FAM-GCTGCACTGTGGG
     TAGGC-3^0 and reverse 5^0 -GGGCGTCGGCACCTTC-3^0.
   
   
   
   


3. Capillary electrophoresis.


4. GeneMapper®software v3.7.

2.5 Sequencing 1. Sequencing primers:

• PRKAA1 primers: forward 50 -ATCACCAGG
  ATCCTTTGGCA-3^0 and reverse 5^0 -TGCTTTCCTTA-
  CACCTTGGTG-3^0.


• PRKAA2 primers: forward 5^0 -GCTGCACTGTGGGTAGG
  C-3^0 and reverse 5^0 -GGGCGTCGGCACCTTC-3^0.

2. Cell lysis buffer: 4% Tween 20,100μg/ml proteinase K in
   H 2 O.


3. DNA polymerase: Phire Hot Start II DNA Polymerase and 5
   reaction buffer (Thermo Scientific).


4. Tris-acetate-EDTA (TAE) buffer: 40 mM Tris–HCl, pH 8.3,
   20 mM acetic acid, 1 mM EDTA.


5. Agarose gel: 2% (w/v) agarose in TAE.

3 Methods

3.1 sgRNA Design
and Cloning

Putative target sites can be identified by simply scanning the region

of the particular genomic location that you want to target (but you

should check your sgRNA design for potential off-target effects) or

by using online bioinformatical tools dedicated to sgRNA design.

(SeeNote 3).

CRISPR-Cas9 For Human AMPK 175