DNA Protocols

Preparing Chemically Competent E. coli Cells

1. Inoculate 5-10mL LB with Top10 E. coli culture at 37°C shaking over night
2. Subculture 1mL of bacteria into 50mL LB at 37oC shaking until OD₆₀₀ is 0.4-0.6 (~2.5 hr)
3. Centrifuge the subculture at max for 20 min at 4°C
4. Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)

Rehydrating Registry DNA

1. Add ddH2O (10μL) to appropriate well (will turn orange)
2. Incubate at room temperature for 10 min
3. Transform cells with DNA (1μL)

Bacterial Transformation

1. Thaw 100μL of competent cells on ice right before use (do not thaw completely)
2. Add DNA (max 20μL) to cells, flick to mix, and ice for 30 min
3. Heat shock 2 min at 42°C
4. Ice 5 min
5. Add 250μL LB medium to each tube
6. Incubate 30-60 min at 37°C shaking (Kan is always 1h+)
7. Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL
8. Resuspend, spread 50μL on antibiotic plate

Plasmid Miniprep (E. coli)

• Resuspension (P1): 50mM Tris HCl (pH 8), 10mM EDTA, 100μg/mL RNase A (keep on ice, 4°C)
• Lysis (P2): 200mM NaOH, 1% SDS
• Precipitation (P3): 3M K Acetate (pH 5.5)

1. Transfer O/N to 2mL tube and pellet culture (spin at 14,000 rpm for 2 mins). Dump supernatant, and repeat for the rest of O/N.
2. Discard supernatant, resuspend pellet in P1 (300μL)
3. Quickly: Add P2 (300 μL), invert gently 2-3x. Add P3 (300 μL), invert gently 2-3x
4. Centrifuge at 14,000 rpm, 10mins, room temp
5. Without disturbing pellet, transfer supernatant to clean 1.5mL tube. Add isopropanol (650 μL). Wait 10 mins, room temp
6. Spin at 14,000 rpm for 10 mins at 4oC
7. Discard supernatant, add cold 70% etOH (500 μL)
8. Centrifuge at 14,000 rpm for 5 mins at 4oC
9. Carefully discard supernatant, dry pellet upside down
10. Resuspend in sterile ddH2O (~30 μL)

Plasmid Miniprep (GenElute Kit)

1. Pellet cells from O/N culture
2. Discard supernatant, and resuspend with Resuspension solution (200 μL)
3. Add Lysis Solution (300 μL), immediately invert 6-8x (gently) until mixture becomes clear. Do not allow reaction to exceed 5 mins
4. Add Neutralization Solution (350 μL), invert 4-6x (gently). Pellet in centrifuge at max, 10 mins. If supernatant contains a large amount of floating particles, respin
5. Meanwhile, prepare binding column. Insert column into a microcentrifuge tube and add Column Prep Solution (500 μL). Spin at 13,000 rpm for 1 min, Discard flow through
6. Transfer the clear lysate (from step 4) to the column, and spin at 13,000 rpm, 1 min. Discard flow through
7. Add Wash Solution (750 μL) to column. Spin at 13,000 rpm, 1 min. Discard flow through and spin again at max, 2 mins
8. Transfer column a fresh collection tube and add ddH2O (~50 μL). Let sit 2 mins. Spin at 13,000 rpm, 1 min. Sample now ready for nano-drop
Restriction Digest

Separately, into labeled tubes for Insert and Vector, add the following reagents:

• 1μg DNA
• 5μL of CutSmart Buffer
• 0.5 to 1μL enzyme 1
• 0.5 to 1μL enzyme 2
• ddH2O to bring total vol to 50μL
Incubate tubes in 37°C water bath, 1 hr Deactivate enzymes: place insert tube on 82°C heatblock, 20 mins. Preform Antarctic phosphatase treatment on Vector tube
Antarctic Phosphatase Treatment
1. To Vector tube from restriction digest, add 10x Antarctic Phosphatase Buffer (5 μL), ddH2O (9 μL), and Antarctice phosphatase (1 μL)
2. Place tube in 37°C water bath, 30 min
3. Transfer to 82°C heatblock, 20min
Ligation (Using KAPA Quick Ligase Kit)
• 10 μL Ligase Buffer
• 50ng Vector DNA (from digest)
• 1:3 to 1:5 [Vector:Insert] molar ratio of Insert DNA (from digest)
• 1 μL Enzyme (Quick Ligase)
• ddH2O to bring total vol to 20 μL
Wait 5 minutes after adding the Quick Ligase enzyme, then transform cells